CN111593010B - Lactobacillus plantarum and method for producing fermented feed by using same - Google Patents
Lactobacillus plantarum and method for producing fermented feed by using same Download PDFInfo
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- 240000006024 Lactobacillus plantarum Species 0.000 title claims abstract description 68
- 235000013965 Lactobacillus plantarum Nutrition 0.000 title claims abstract description 68
- 229940072205 lactobacillus plantarum Drugs 0.000 title claims abstract description 68
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 26
- 238000004321 preservation Methods 0.000 claims abstract description 15
- 238000000855 fermentation Methods 0.000 claims description 60
- 230000004151 fermentation Effects 0.000 claims description 60
- 239000001963 growth medium Substances 0.000 claims description 40
- 239000007788 liquid Substances 0.000 claims description 38
- 239000007787 solid Substances 0.000 claims description 34
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 22
- 238000012258 culturing Methods 0.000 claims description 12
- 239000004310 lactic acid Substances 0.000 claims description 11
- 235000014655 lactic acid Nutrition 0.000 claims description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
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- 238000000034 method Methods 0.000 claims description 8
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- 239000001888 Peptone Substances 0.000 claims description 5
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- 238000002360 preparation method Methods 0.000 claims description 4
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- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
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- 241001465754 Metazoa Species 0.000 description 4
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Natural products OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 4
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
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- 235000019764 Soybean Meal Nutrition 0.000 description 2
- 235000019752 Wheat Middilings Nutrition 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
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- 150000001875 compounds Chemical class 0.000 description 2
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
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- 229920001817 Agar Polymers 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
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- 150000001412 amines Chemical class 0.000 description 1
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- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
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- 230000002708 enhancing effect Effects 0.000 description 1
- 230000003031 feeding effect Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
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- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
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Classifications
-
- 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
- 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
- A23K10/10—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
- 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
- A23K10/33—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from molasses
-
- 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
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
-
- 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
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- 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
- A23K20/26—Compounds containing phosphorus
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- 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
Abstract
The invention belongs to the technical field of bioengineering, and provides a lactobacillus plantarum and a method for producing fermented feed by using the same, wherein the lactobacillus plantarum has a strain code of YJY19-08 and a preservation number of CGMCC No. 18401; the strain has strong growth capacity and acid production capacity, the fermented feed product has long storage time and high survival rate of lactobacillus plantarum, can obviously improve the feed intake of livestock and poultry, promote the digestion and absorption of nutrition, reduce the feed conversion ratio, and has high utilization value.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and provides a lactobacillus plantarum strain and a method for producing fermented feed by using the same.
Background
The feed added with antibiotics can improve the growth performance of piglets and reduce diarrhea rate and death rate. At present, feed additives containing antibiotics are commonly used in Chinese farms to achieve the purposes of preventing diseases and promoting growth. This will increase the risk of drug resistance by pathogenic bacteria in animals and allow the transfer of these resistant bacteria and genes to humans. In 2006, 1.1, antibiotic additives are completely prohibited from being applied to animal feed in European Union, and other countries also increase the use limit of antibiotics. The search for safe, efficient and environment-friendly substitutes is a problem to be solved urgently at present.
The fermented feed can be regarded as a compound of a viable bacteria preparation and a fermentation substrate, plant agricultural and sideline products are used as main raw materials (substrates), and partial macromolecular substances such as polysaccharide, protein and fat are degraded through the metabolic action of microorganisms to generate small molecular substances such as organic acid, soluble polypeptide and the like, so that the biological feed or feed raw material with rich nutrition, good palatability and high viable bacteria content is formed.
The lactobacillus plantarum belongs to lactobacillus and gram-positive bacteria, the lactobacillus is used for fermenting the feed, the feed is fed to livestock and poultry fermented feed, the microecological balance in intestinal tracts of the livestock and poultry can be maintained to play a role, the concentration of harmful gases such as ammonia gas, hydrogen sulfide and the like in a culture room can be reduced, and the feed has the advantages of preventing and treating diseases, enhancing the immunity of organisms, improving the growth performance, having no toxic or side effect, drug resistance, residue, promoting growth, increasing the weight and the like. The lactobacillus is one of the feed-grade probiotic strains which are published by the ministry of agriculture in China and can be directly fed to animals and are allowed to be used, and the fermented feed for feeding the livestock has good antibacterial performance and stability, has good probiotic effect on animals and human beings, and has wide prospect when being used for livestock breeding.
Therefore, screening better strains with the function of degrading organic amines from the nature becomes one of the important tasks of researchers.
Disclosure of Invention
Aiming at the defects of the technology, the invention provides a lactobacillus plantarum and a method for producing fermented feed by using the lactobacillus plantarum, wherein the lactobacillus plantarum has a strain code of YJY19-08 and a preservation number of CGMCC No. 18401; the strain has strong growth capacity and acid production capacity, the fermented feed product has long storage time and high survival rate of lactobacillus plantarum, can obviously improve the feed intake of livestock and poultry, promote the digestion and absorption of nutrition, reduce the feed conversion ratio, and has high utilization value.
The specific technical scheme provided by the invention is as follows:
the invention obtains a new Lactobacillus plantarum (Lactobacillus plantarum) with the biological preservation number of CGMCC No.18401 and the name of YJY19-08, and the specific obtaining method is as follows:
the bacterial strain is a high-efficiency lactobacillus plantarum bacterial strain obtained by sampling fresh excrement of a certain pig farm factory in Shandong and performing primary screening and secondary screening, and the screening process is as follows: taking 1g of fresh pig manure sample by aseptic operation, diluting the fresh pig manure sample into uniform diluent of 100 times and 1000 times by using aseptic normal saline, taking 0.1mL of diluent, coating the diluent in a solid MRS culture medium containing calcium carbonate, culturing at the constant temperature of 35 ℃ for 48h, selecting a single colony capable of generating a transparent ring, performing streak culture, selecting the single colony, preserving at the low temperature in a glycerol culture medium,
inoculating the strain preserved at low temperature into 200mL of liquid MRS culture medium, culturing at 32 ℃ for 48h, and detecting the content, pH and acid yield of viable bacteria in fermentation liquor; the lactobacillus strain with highest activity and strongest acid production capability is sent to a microorganism research institute of Chinese academy of sciences for identification, and is identified as lactobacillus plantarum according to a sequencing result, and the morphological characteristics of the strain are as follows: the strain is round in a solid MRS culture medium, the bacterial colony is small, the edge is neat, the outer surface of the bacterial body is milky white, the bulge is bright, and the bacterial colony in the culture medium is round and flat. Observing under a microscope, wherein the strain is rod-shaped, sending the strain to a institute of microbiology of Chinese academy of sciences for 16S rDNA sequencing, wherein the sequence is shown as Seq ID No:1, and identifying the strain as Lactobacillus plantarum (Lactobacillus plantarum) according to a sequencing result;
the inventor further performs biological preservation, and the biological preservation number is CGMCC No. 18401;
the inventor carries out related acid production experiments on the strain, and the result shows that the lactobacillus plantarum has the advantages of higher growth speed and high acid production, and the bacterial yield can reach 2.8 x 10 after 24 hours of fermentation at 32 DEG C 9 CFU/mL, the acid yield can reach 12.3 g/L.
Therefore, the inventor applies the lactobacillus plantarum fermentation liquid to the production of fermented feed, and respectively prepares lactobacillus plantarum fermentation liquid and lactobacillus plantarum solid fermentation products:
the preparation method of the lactobacillus plantarum fermentation liquor comprises the following specific steps:
(1) activating strains: inoculating 10-30 μ L of frozen Lactobacillus plantarum in 200mL MRS-containing liquid sterile culture medium, and performing shake culture at 28-38 deg.C and 180rpm at 160-;
(2) preparing liquid seeds: 200mL of the activated strain is transferred to a liquid sterile culture medium containing 1000mL of MRS, and the liquid seed is obtained by shaking culture for 24h at the temperature of 28-38 ℃ and the rpm of 160-180-;
(3) fermenting in a seeding tank: inoculating the prepared liquid seeds into a 20L seed tank with 70% liquid containing amount according to the inoculation amount of 5% -10% v/v for expanded culture, wherein the specific culture conditions are as follows: keeping the temperature of the tank at 25-28 ℃, the pressure of the tank at 30-40kpa, the stirring speed at 100-: 0.2-0.4, pH 3.4-3.6, fermenting for 76-96 hours; the culture medium in the seeding tank is MRS liquid sterile culture medium;
(4) fermentation in a production tank: inoculating the seed solution into culture medium of 70% production tank according to inoculum size of 2-10% v/v, culturing, and making the thallus number reach 5.0 x 10 9 More than mL, taking the culture solution out of the tank after fermentation is finished to obtain lactobacillus plantarum fermentation liquor;
the culture medium in the production tank in the step (4) comprises the following components: 88g of brown sugar, 1.1g of yeast extract, 1.1g of peptone, 0.35g of monopotassium phosphate, 105g of molasses and 1.85g of sodium hydroxide, wherein the pH is natural, and distilled water is added to the solution to reach the constant volume of 1L.
The obtained fermentation liquor can be directly used as a feed additive, and the fermentation liquor and the conventional common solid feed are mixed according to the proportion of 1: 9-1: 19, mixing the components in a weight ratio and directly feeding the mixture;
in addition, the fermentation liquor can be used as the initial material to carry out solid fermentation to obtain a corresponding solid fermentation product, and the specific steps are as follows:
(5) inoculation and fermentation: inoculating the obtained lactobacillus plantarum fermentation liquor into a solid culture medium according to 0.01-0.1 ml per gram, uniformly mixing, and hermetically fermenting for 72 hours at 25-32 ℃ to obtain a solid fermentation product;
the solid culture medium comprises the following components in percentage by weight: 15% of wheat middling, 35% of corn flour, 15% of wheat bran and 7% of soybean meal, and adding a proper amount of water to adjust the water content to 40% -50%;
the inventor carries out related detection aiming at the content of lactobacillus in liquid seeds, the pH value and the lactic acid content of the liquid seeds, the content of lactobacillus in fermentation liquor and the content of lactobacillus in solid fermented feed in the fermentation process, and the specific process and the result are as follows:
taking 1mL of the lactobacillus plantarum fermentation broth obtained in the step (2), and diluting the fermentation broth in a gradient manner by 10 8 The viable bacteria content of the product is 2.8 x 10 measured after the product is coated in a solid MRS culture medium and cultured for 48 hours at 32 DEG C 9 CFU/mL；
Centrifuging the lactobacillus plantarum fermentation liquor obtained in the step (2) to measure the pH and the lactic acid content, wherein the pH is 3.0-3.5, the lactic acid content is 10.0-15.0g/L, the pH is preferably controlled to be 3.27, and the lactic acid content is 12.3 g/L;
taking 1mL of the lactobacillus plantarum fermentation broth obtained in the step (4), and diluting the fermentation broth in a gradient manner by 10 8 The viable bacteria content of the product is 5.2 x 10 measured after the product is coated in a solid MRS culture medium and cultured for 48 hours at 32 DEG C 9 CFU/mL；
Taking 1g of the solid fermentation product obtained in the step (5), and diluting by gradient 10 8 Coating the mixture in solid MRS culture medium, culturing at 32 deg.C for 48 hr, counting lactobacillus colonies, and calculating to obtain viable bacteria content of 4.6 × 10 9 CFU/g; the residual bacterial count after 7 days of storage at 28 ℃ was 2.76 x 10 9 CFU/g; the residual bacterial count after 14d was 1.58 x 10 9 CFU/g; the residual bacterial amount after 20 days is 1.03 x 10 9 CFU/g, survival rate 23%;
the bacterial quantity of 2d of conventional lactic acid bacteria solid fermentation in the prior art is about 20-50 hundred million CFU/g, and the bacterial quantity of 2d of bacterial strain fermentation obtained by the invention can be stabilized at about 45-50 hundred million CFU/g and is at a higher level, which shows that the bacterial strain obtained by the invention has higher activity and good fermentation performance.
According to specific experiments, the beneficial effects of the invention are verified as follows:
1. the lactobacillus plantarum has the advantages of high growth speed and high acid yield, and the bacterial yield can reach 2.8 x 10 after 24 hours of fermentation at 32 DEG C 9 cfu/mL, the acid yield can reach 12.3 g/L;
2. the lactobacillus plantarum is used for preparing fermented feed, namely the solid fermented product obtained in the step (5), and the bacterial quantity can reach 4.5 x 10 after 72 hours of fermentation at the temperature of 32 DEG C 9 cfu/g, which can effectively inhibit the growth of other microorganisms in the fermented feed when being directly fed as the feed, decompose macromolecular protein and fat in a solid culture medium, and improve the nutritional level;
3. the fermented feed prepared by the lactobacillus plantarum is beneficial to storage, the survival rate of the lactobacillus plantarum after being preserved for 20 days at normal temperature in a sealing manner can reach more than 20%, and compared with the conventional lactobacillus, the lactobacillus plantarum provided by the invention has the advantages of higher survival rate, higher activity and strong tolerance.
The information of the storage is stored in the storage device,
preservation time: 8/20/2019
The name of the depository: china general microbiological culture Collection center
The preservation number is: CGMCC No.18401
The address of the depository: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
Classification nomenclature Lactobacillus plantarum (Lactobacillus plantarum)
Detailed Description
The present invention will be described in further detail with reference to the following examples, but it should not be construed that the scope of the above subject matter is limited to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention, except for the specific description, the following examples all adopt the conventional prior art to complete the strains adopted in the following examples are the strains with the preservation number of CGMCC No. 18401.
Example 1
A new Lactobacillus plantarum (Lactobacillus plantarum) with the biological preservation number of CGMCC No.18401 is named as YJY19-08, and the specific obtaining method comprises the following steps:
(1) taking fresh excrement from a piggery of a pig farm, weighing 1g of fresh pig excrement sample in a sterile operation, and diluting the fresh pig excrement sample into uniform diluent which is 100 times and 1000 times of sterile normal saline;
(2) coating 0.1mL of diluent in MRS selective medium containing calcium carbonate, and culturing at constant temperature of 35 ℃ for 48 h;
(3) selecting a single colony capable of generating a transparent ring, performing streak culture, and selecting the single colony to be preserved in a glycerol culture medium at a low temperature;
(4) inoculating the strain preserved at low temperature into 200mL of liquid MRS culture medium, culturing for 48h at 32 ℃, and detecting the content of viable bacteria, pH and acid yield in fermentation liquor;
(5) the Lactobacillus strain with the highest activity and the strongest acid production capacity is sent to a microbiological research institute of the Chinese academy of sciences to be identified, and is identified as Lactobacillus plantarum (Lactobacillus plantarum) according to a sequencing result. The morphological characteristics of the strain are as follows: the strain is round in a solid MRS culture medium, the bacterial colony is small, the edge is neat, the outer surface of the bacterial body is milky white, the bulge is bright, and the bacterial colony in the culture medium is round and flat. Observed under a microscope, the rod-shaped. The strain is sent to the institute of microbiology of Chinese academy of sciences for 16S rDNA sequencing, the sequence is shown as SeqID No:1, and the strain is identified as Lactobacillus plantarum (Lactobacillus plantarum) according to the sequencing result;
the inventor further carries out biological preservation, the biological preservation number is CGMCC No.18401, and the biological preservation is detected to be in a survival state;
the inventor carries out related acid production experiments on the strain, and the result shows that the lactobacillus plantarum has the advantages of higher growth speed and high acid production, and the bacterial yield can reach 2.8 x 10 after 24 hours of fermentation at 32 DEG C 9 The acid yield can reach 12.3g/L for cfu/mL.
In the process:
liquid MRS medium: 10g of peptone, 10g of beef extract, 5g of yeast powder, 15g of glucose, 5g of sodium acetate, 2g of citric acid diamine, 2g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate and 0.05g of manganese sulfate, wherein the pH value is natural, 1000mL of distilled water is used, and the sterilization is carried out at 121 ℃ for 20 min.
MRS selection medium: 10g of peptone, 10g of beef extract, 5g of yeast powder, 15g of glucose, 5g of sodium acetate, 2g of citric acid diamine, 2g of dipotassium phosphate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 7.5g of calcium carbonate and 15g of agar, wherein the pH value is natural, 1000mL of distilled water is used, and the beef is sterilized for 20min at 121 ℃.
Example 2
Growth performance and acid production performance of the strains in the laboratory were determined:
2.1 liquid Strain preparation
The strain is inoculated into a liquid MRS culture medium and placed at 32 ℃,160-180rpm for shaking culture until the logarithmic phase.
Inoculating the culture solution into liquid MRS culture medium according to 5% (v/v), and culturing at 32 deg.C for 48h to obtain liquid strain.
2.2 detection of bacterial load
Taking 1mL of the liquid strain, and diluting by gradient 10 8 Coating on solid MRS culture medium, culturing at 32 deg.C for 48 hr, counting lactobacillus colonies, and counting viable bacteria number in liquid strain to 2.9 × 10 9 CFU/g。
2.3 acid yield detection
And (3) taking the seed liquid, and performing centrifugal measurement on the pH and the lactic acid content, wherein the pH is 3.53, and the lactic acid content is 10.5 g/L.
Example 3
The specific steps for preparing the lactobacillus plantarum liquid fermentation product and the solid fermentation product are as follows:
(1) activating strains: inoculating 10-30 μ L of frozen Lactobacillus plantarum in 200mL MRS-containing liquid sterile culture medium, and performing shake culture at 35 deg.C and 160rpm to logarithmic phase;
(2) preparing liquid seeds: transferring 200mL of the activated strain into a sterile liquid culture medium containing 1000mL of MRS, and performing shaking culture at 35 ℃ and 160rpm for 24 h;
(3) fermenting in a seeding tank: inoculating the prepared liquid seeds into a 20L seed tank with 70% liquid loading according to the inoculation amount of 5% -10% v/v for expanded culture, wherein the specific culture conditions are as follows: keeping the tank temperature at 25-28 ℃, the tank pressure at 30-40kpa, the stirring speed at 120r/min, and the ventilation volume at 1: 0.2-0.4, pH 3.4-3.6, fermenting for 76-96 hours; the culture medium in the seeding tank is MRS liquid sterile culture medium;
(4) fermentation in a production tank: inoculating the seed solution into culture medium of 70% production tank according to inoculum size of 2-10% v/v, culturing, and making the thallus number reach 5.0 x 10 9 More than CFU/mL, taking the culture solution out of the tank after fermentation is finished to obtain lactobacillus plantarum fermentation liquor;
the culture medium in the production tank in the step (4) comprises the following components: 88g of brown sugar, 1.1g of yeast extract, 1.1g of peptone, 0.35g of monopotassium phosphate, 105g of molasses and 1.85g of sodium hydroxide, wherein the pH is natural, and distilled water is added to the solution to reach the constant volume of 1L.
The obtained fermentation liquor can be directly used as a feed additive, and the fermentation liquor and the conventional common solid feed are mixed according to the proportion of 1: 9-1: 19, mixing the components in a weight ratio and directly feeding the mixture;
in addition, the fermentation liquor can be used as the initial material to carry out solid fermentation to obtain a corresponding solid fermentation product, and the specific steps are as follows:
(5) inoculation and fermentation: inoculating the obtained lactobacillus plantarum fermentation liquor into a solid culture medium according to 0.01-0.10 ml per gram, uniformly mixing, and hermetically fermenting for 72 hours at 28 ℃ to obtain a solid fermentation product;
the solid culture medium comprises the following components in percentage by weight: 15% of wheat middling, 35% of corn flour, 15% of wheat bran and 7% of soybean meal, and adding a proper amount of water to adjust the water content to 40% -50%;
taking 1mL of the lactobacillus plantarum liquid seeds obtained in the step (2), and diluting the lactobacillus plantarum liquid seeds in a gradient way by 10 8 Spreading in solid MRS culture medium, culturing at 32 deg.C for 48 hr to obtain viable bacteria content of 2.8 x 10 9 CFU/mL；
Centrifuging the lactobacillus plantarum liquid seeds obtained in the step (2) to measure the pH and the lactic acid content, wherein the pH is 3.27, and the lactic acid content is 12.3 g/L;
taking 1mL of lactobacillus plantarum fermentation broth obtained in the step (4), and diluting the fermentation broth in a gradient manner by 10 8 Double-coated solid MRS culture mediumCulturing at 32 deg.C for 48 hr to obtain product with viable bacteria content of 5.2 x 10 9 CFU/mL；
Taking 1g of the solid fermentation product obtained in the step (5), and diluting the solid fermentation product in a gradient way by 10 8 Coating the mixture in solid MRS culture medium, culturing at 32 deg.C for 48 hr, counting lactobacillus colonies, and calculating to obtain viable bacteria content of 4.6 × 10 9 CFU/g。
The nutrient contents of the fermented product before and after fermentation were measured, and the results are shown in Table 1.
TABLE 1 determination of nutrient content of Lactobacillus plantarum fermented feed
The results show that after 10 days of fermentation, the crude protein content remains substantially unchanged and the acid soluble protein content increases significantly. Meanwhile, indexes of crude fat, crude fiber and ash content are obviously reduced. The lactobacillus plantarum strain is shown to have the effects of improving the nutrition structure of the fermented feed and improving the utilization rate of the feed.
Example 4
Fermented feed preservation and nursery pig feeding experiments:
4.1 fermented feed preservation Effect
Storing the solid fermentation product prepared by the method at 28 ℃, periodically observing whether the solid fermentation product is mildewed or not and whether abnormal odor exists or not, and simultaneously sampling and detecting the content of the residual lactobacillus plantarum.
The results showed that the residual bacterial mass after 7 days of storage was 2.76 x 10 9 CFU/g; the residual bacterial count after 14d was 1.58 x 10 9 CFU/g; the residual bacterial amount after 20 days is 1.03 x 10 9 CFU/g, survival 23%. The survival rate of the strain is superior to that of the common lactobacillus plantarum. The fermented feed is stored for 40 days at 28 ℃ without mildewing.
4.2 fermented feed feeding effect
20 fattening pigs of about 50kg with similar weight, same male and female quantity and good health state are selected, and are averagely divided into two groups, namely an experimental group and a control group, wherein each group has 2 repetitions, and each repetition has 5 repetitions. The experiment took 60 days, with one phase every 30 days. The control group was fed with commercial complete feed, and the experimental group was fed with compound feed (fermented feed: commercial complete feed: 1: 9). The results of the experiments at each stage are shown in Table 2.
TABLE 2 influence of Lactobacillus plantarum fermented feed on pig growth performance
Note: a is the experimental group and B is the control group.
The results show that the average daily weight gain of the test group in the first month, the second month and the whole test period are respectively improved by 5.53 percent, 15.34 percent and 10.77 percent compared with the control group; the feed meat of the test group is respectively reduced by 5.24 percent, 13.30 percent and 12.00 percent compared with the control group. The fermented feed prepared on the basis of the lactobacillus plantarum is shown to have the effects of improving the feed intake and reducing the feed conversion ratio, and has higher utilization value.
Sequence listing
<110> Jingbo chemical research institute of yellow river delta Ltd
<120> lactobacillus plantarum and method for producing fermented feed by using same
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<212> DNA
<213> Lactobacillus plantarum (Lactobacillus plantarum)
<400> 1
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tattcaccgc ggcatgctga tccgcgatta ctagcgattc cgacttcatg taggcgagtt 120
gcagcctaca atccgaactg agaatggctt taagagatta gcttactctc gcgagttcgc 180
aactcgttgt accatccatt gtagcacgtg tgtagcccag gtcataaggg gcatgatgat 240
ttgacgtcat ccccaccttc ctccggtttg tcaccggcag tctcaccaga gtgcccaact 300
taatgctggc aactgataat aagggttgcg ctcgttgcgg gacttaaccc aacatctcac 360
gacacgagct gacgacaacc atgcaccacc tgtatccatg tccccgaagg gaacgtctaa 420
tctcttagat ttgcatagta tgtcaagacc tggtaaggtt cttcgcgtag cttcgaatta 480
aaccacatgc tccaccgctt gtgcgggccc ccgtcaattc ctttgagttt cagccttgcg 540
gccgtactcc ccaggcggaa tgcttaatgc gttagctgca gcactgaagg gcggaaaccc 600
tccaacactt agcattcatc gtttacggta tggactacca gggtatctaa tcctgtttgc 660
tacccatact ttcgagcctc agcgtcagtt acagaccaga cagccgcctt cgccactggt 720
gttcttccat atatctacgc atttcaccgc tacacatgga gttccactgt cctcttctgc 780
actcaagttt cccagtttcc gatgcacttc ttcggttgag ccgaaggctt tcacatcaga 840
cttaaaaaac cgcctgcgct cgctttacgc ccaataaatc cggacaacgc ttgccaccta 900
cgtattaccg cggctgctgg cacgtagtta gccgtggctt tctggttaaa taccgtcaat 960
acctgaacag ttactctcag atatgttctt ctttaacaac agagttttac gagccgaaac 1020
ccttcttcac tcacgcggcg ttgctccatc agactttcgt ccattgtgga agattcccta 1080
ctgctgcctc ccgtaggagt ttgggccgtg tctcagtccc aatgtggccg attaccctct 1140
caggtcggct acgtatcatt gccatggtga gccgttaccc caccatctag ctaatacgcc 1200
gcgggaccat ccaaaagtga tagccgaagc catctttcaa gctcggacca tgcggtccaa 1260
gttgttatgc ggtattagca tctgtttcca ggtgttatcc cccgcttctg ggcaggtttc 1320
ccacgtgtta ctcaccagtt cgccactcac tcaaatgtaa atcatgatgc aagcaccaat 1380
ca 1382
Claims (4)
1. Lactobacillus plantarum (f)Lactobacillus plantarum) The biological preservation number is CGMCC No.18401, and the name is YJY 19-08;
the nucleotide sequence of the 16S rDNA of the lactobacillus plantarum is shown as Seq ID No. 1.
2. A method for producing fermented feed using lactobacillus plantarum according to claim 1, characterized in that: the preparation method of the lactobacillus plantarum fermentation liquor comprises the following specific steps:
(1) activating strains: inoculating 10-30 μ L of frozen Lactobacillus plantarum in 200mL MRS-containing liquid sterile culture medium, and performing shake culture at 28-38 deg.C and 180rpm at 160-;
(2) preparing liquid seeds: 200mL of the activated strain is transferred to a liquid sterile culture medium containing 1000mL of MRS, and the liquid seed is obtained by shaking culture for 24h at the temperature of 28-38 ℃ and the rpm of 160-180-;
(3) fermenting in a seeding tank: inoculating the prepared liquid seeds into a 20L seed tank with 70% liquid containing amount according to the inoculation amount of 5% -10% v/v for expanded culture, wherein the specific culture conditions are as follows: keeping the tank temperature at 25-28 ℃, the tank pressure at 30-40kpa, the stirring speed at 100-: 0.2-0.4, pH 3.4-3.6, fermenting for 76-96 hours; the culture medium in the seeding tank is MRS liquid sterile culture medium;
(4) fermentation in a production tank: inoculating the seed solution into culture medium of 70% production tank according to inoculum size of 2-10% v/v, culturing, and allowing the number of thallus to reach 5.0 x 10 9 More than mL, taking the culture solution out of the tank after fermentation is finished to obtain lactobacillus plantarum fermentation liquor;
the culture medium in the production tank in the step (4) comprises the following components: 88g of brown sugar, 1.1g of yeast extract, 1.1g of peptone, 0.35g of monopotassium phosphate, 105g of molasses and 1.85g of sodium hydroxide, wherein the pH is natural, and distilled water is added to the solution to reach the constant volume of 1L.
3. A method of producing fermented feed with lactobacillus plantarum according to claim 2, characterized in that: the obtained fermentation liquor is directly used as a feed additive, and the fermentation liquor and conventional common solid feed are mixed according to the proportion of 1: 9-1: 19 by weight ratio and directly feeding.
4. A method of producing fermented feed with lactobacillus plantarum according to claim 2, characterized in that: and (3) centrifuging the liquid seeds obtained in the step (2) to measure the pH and the lactic acid content, wherein the pH is 3.0-3.5, and the lactic acid content is 10.0-15.0 g/L.
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| CN104946564A (en) * | 2015-06-18 | 2015-09-30 | 郑州大学 | Lactobacillus plantarum and application thereof in low temperature ensiling |
| CN105483050A (en) * | 2015-12-31 | 2016-04-13 | 北京英惠尔生物技术有限公司 | Lactobacillus plantarum strain and application thereof in fermented feed |
| CN107177522A (en) * | 2016-03-09 | 2017-09-19 | 北京大伟嘉生物技术股份有限公司 | One plant height activity forage plant lactobacillus and its cultural method and application |
| CN110982739A (en) * | 2019-12-05 | 2020-04-10 | 四川省草原科学研究院 | Lactobacillus plantarum for high-moisture oat silage and application thereof |
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| CN104946564A (en) * | 2015-06-18 | 2015-09-30 | 郑州大学 | Lactobacillus plantarum and application thereof in low temperature ensiling |
| CN105483050A (en) * | 2015-12-31 | 2016-04-13 | 北京英惠尔生物技术有限公司 | Lactobacillus plantarum strain and application thereof in fermented feed |
| CN107177522A (en) * | 2016-03-09 | 2017-09-19 | 北京大伟嘉生物技术股份有限公司 | One plant height activity forage plant lactobacillus and its cultural method and application |
| CN110982739A (en) * | 2019-12-05 | 2020-04-10 | 四川省草原科学研究院 | Lactobacillus plantarum for high-moisture oat silage and application thereof |
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