CN113186148A - Peptide for improving bifidobacterium longum proliferation efficiency and application thereof - Google Patents
Peptide for improving bifidobacterium longum proliferation efficiency and application thereof Download PDFInfo
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- CN113186148A CN113186148A CN202110600881.0A CN202110600881A CN113186148A CN 113186148 A CN113186148 A CN 113186148A CN 202110600881 A CN202110600881 A CN 202110600881A CN 113186148 A CN113186148 A CN 113186148A
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- bifidobacterium longum
- nitrogen source
- peptide
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- 241001608472 Bifidobacterium longum Species 0.000 title claims abstract description 82
- 229940009291 bifidobacterium longum Drugs 0.000 title claims abstract description 82
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 53
- 230000035755 proliferation Effects 0.000 title claims abstract description 27
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 118
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 59
- 238000000034 method Methods 0.000 claims abstract description 26
- 239000001963 growth medium Substances 0.000 claims abstract description 25
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 13
- 238000012258 culturing Methods 0.000 claims abstract description 6
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 17
- 239000000843 powder Substances 0.000 claims description 17
- 239000012138 yeast extract Substances 0.000 claims description 13
- 108010076119 Caseins Proteins 0.000 claims description 11
- 239000005018 casein Substances 0.000 claims description 11
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 11
- 235000021240 caseins Nutrition 0.000 claims description 11
- 239000012528 membrane Substances 0.000 claims description 9
- 238000000108 ultra-filtration Methods 0.000 claims description 9
- 239000002609 medium Substances 0.000 claims description 8
- 235000018102 proteins Nutrition 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 102000004142 Trypsin Human genes 0.000 claims description 7
- 108090000631 Trypsin Proteins 0.000 claims description 7
- 239000001110 calcium chloride Substances 0.000 claims description 7
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 7
- 229910052564 epsomite Inorganic materials 0.000 claims description 7
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 7
- 229920000136 polysorbate Polymers 0.000 claims description 7
- 239000012588 trypsin Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- QIJRTFXNRTXDIP-UHFFFAOYSA-N (1-carboxy-2-sulfanylethyl)azanium;chloride;hydrate Chemical compound O.Cl.SCC(N)C(O)=O QIJRTFXNRTXDIP-UHFFFAOYSA-N 0.000 claims description 6
- 239000007836 KH2PO4 Substances 0.000 claims description 6
- 108091005804 Peptidases Proteins 0.000 claims description 6
- 239000004365 Protease Substances 0.000 claims description 6
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 6
- 229940040526 anhydrous sodium acetate Drugs 0.000 claims description 6
- 235000015190 carrot juice Nutrition 0.000 claims description 6
- 229960001305 cysteine hydrochloride Drugs 0.000 claims description 6
- YXVFQADLFFNVDS-UHFFFAOYSA-N diammonium citrate Chemical compound [NH4+].[NH4+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O YXVFQADLFFNVDS-UHFFFAOYSA-N 0.000 claims description 6
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 6
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 6
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 6
- WQZGKKKJIJFFOK-DVKNGEFBSA-N alpha-D-glucose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-DVKNGEFBSA-N 0.000 claims description 5
- 108010079058 casein hydrolysate Proteins 0.000 claims description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- 239000011347 resin Substances 0.000 claims description 5
- 229920005989 resin Polymers 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 230000009849 deactivation Effects 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 230000001376 precipitating effect Effects 0.000 claims description 3
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 3
- 230000012010 growth Effects 0.000 abstract description 14
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- 244000005700 microbiome Species 0.000 abstract description 5
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- 239000000306 component Substances 0.000 description 25
- 102000011632 Caseins Human genes 0.000 description 9
- 229940041514 candida albicans extract Drugs 0.000 description 8
- 239000001888 Peptone Substances 0.000 description 7
- 108010080698 Peptones Proteins 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 235000019319 peptone Nutrition 0.000 description 7
- 102000004196 processed proteins & peptides Human genes 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 241000186000 Bifidobacterium Species 0.000 description 6
- 239000002028 Biomass Substances 0.000 description 6
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- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- 102000007079 Peptide Fragments Human genes 0.000 description 3
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- 150000001413 amino acids Chemical class 0.000 description 3
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
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- VRYALKFFQXWPIH-PBXRRBTRSA-N (3r,4s,5r)-3,4,5,6-tetrahydroxyhexanal Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)CC=O VRYALKFFQXWPIH-PBXRRBTRSA-N 0.000 description 2
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 2
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 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
- 238000007696 Kjeldahl method Methods 0.000 description 2
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-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
- 238000002835 absorbance Methods 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- PMMURAAUARKVCB-UHFFFAOYSA-N alpha-D-ara-dHexp Natural products OCC1OC(O)CC(O)C1O PMMURAAUARKVCB-UHFFFAOYSA-N 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
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- 239000003480 eluent Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 239000000413 hydrolysate Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 description 2
- 235000011009 potassium phosphates Nutrition 0.000 description 2
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- MINDHVHHQZYEEK-UHFFFAOYSA-N (E)-(2S,3R,4R,5S)-5-[(2S,3S,4S,5S)-2,3-epoxy-5-hydroxy-4-methylhexyl]tetrahydro-3,4-dihydroxy-(beta)-methyl-2H-pyran-2-crotonic acid ester with 9-hydroxynonanoic acid Natural products CC(O)C(C)C1OC1CC1C(O)C(O)C(CC(C)=CC(=O)OCCCCCCCCC(O)=O)OC1 MINDHVHHQZYEEK-UHFFFAOYSA-N 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 1
- 206010010774 Constipation Diseases 0.000 description 1
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 description 1
- 101000993347 Gallus gallus Ciliary neurotrophic factor Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 1
- QGOQADGTOYSTCH-UHFFFAOYSA-N OS(O)(=O)=O.C1=CC=C2C=C3C(=O)CC=CC3=CC2=C1 Chemical compound OS(O)(=O)=O.C1=CC=C2C=C3C(=O)CC=CC3=CC2=C1 QGOQADGTOYSTCH-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- VLSOAXRVHARBEQ-UHFFFAOYSA-N [4-fluoro-2-(hydroxymethyl)phenyl]methanol Chemical compound OCC1=CC=C(F)C=C1CO VLSOAXRVHARBEQ-UHFFFAOYSA-N 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 229960004050 aminobenzoic acid Drugs 0.000 description 1
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- KLOIYEQEVSIOOO-UHFFFAOYSA-N carbocromen Chemical compound CC1=C(CCN(CC)CC)C(=O)OC2=CC(OCC(=O)OCC)=CC=C21 KLOIYEQEVSIOOO-UHFFFAOYSA-N 0.000 description 1
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- DDHVILIIHBIMQU-YJGQQKNPSA-L mupirocin calcium hydrate Chemical compound O.O.[Ca+2].C[C@H](O)[C@H](C)[C@@H]1O[C@H]1C[C@@H]1[C@@H](O)[C@@H](O)[C@H](C\C(C)=C\C(=O)OCCCCCCCCC([O-])=O)OC1.C[C@H](O)[C@H](C)[C@@H]1O[C@H]1C[C@@H]1[C@@H](O)[C@@H](O)[C@H](C\C(C)=C\C(=O)OCCCCCCCCC([O-])=O)OC1 DDHVILIIHBIMQU-YJGQQKNPSA-L 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 229940055726 pantothenic acid Drugs 0.000 description 1
- 235000019161 pantothenic acid Nutrition 0.000 description 1
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- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 235000008160 pyridoxine Nutrition 0.000 description 1
- 239000011677 pyridoxine Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 229960002477 riboflavin Drugs 0.000 description 1
- 235000019192 riboflavin Nutrition 0.000 description 1
- 239000002151 riboflavin Substances 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4732—Casein
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- Engineering & Computer Science (AREA)
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- Genetics & Genomics (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a peptide for improving the multiplication efficiency of bifidobacterium longum and application thereof, belonging to the technical field of microorganisms. The invention aims to solveThe method for improving the proliferation efficiency of the bifidobacterium longum is provided due to the problem that the proliferation of the bifidobacterium longum is influenced by low utilization rate of a nitrogen source, and the bifidobacterium longum is cultured by using hydrophilic peptide with the molecular weight of 1500-2000 Da and rich in proline as the nitrogen source of a culture medium. The method is used for culturing Bifidobacterium longum, and fermentation liquid OD is obtained at a nitrogen source culture concentration of 4g/L600Higher than 2.5 times of other nitrogen sources, the generation time is less than 2.1h, and the utilization rate of the nitrogen source is higher than 35%. The method can remarkably improve the utilization efficiency of the nitrogen source and effectively reduce the waste of the nitrogen source while realizing the high-efficiency promotion of the growth of the strains.
Description
Technical Field
The invention relates to a peptide for improving the multiplication efficiency of bifidobacterium longum and application thereof, belonging to the technical field of microorganisms.
Background
Bifidobacteria (Bifidobacteria) are probiotics existing in human and animal intestinal tracts, are one of normal intestinal flora, and are physiological bacteria influencing the physiological environment of the digestive tract, and the currently determined Bifidobacteria have the efficacies of resisting cancers, regulating immunity, preventing constipation and the like. However, the currently commercialized bifidobacterium strains have few species, mainly because the preparation of bifidobacterium is very difficult, and the nitrogen source is one of the key factors for restricting the effective proliferation of the bifidobacterium.
At present, the culture optimization of bifidobacterium longum at home and abroad is mainly to screen high-quality nitrogen sources through single-factor tests and orthogonal tests, and the nitrogen sources mainly comprise various nitrogen sources such as yeast extract powder, soybean peptone, tryptone, fishbone peptone and the like. However, previous studies found that even the addition of 0.25% yeast extract did not stimulate the growth of bifidobacterium longum ATCC 15708, even though it was a source of nitrogen for yeast extract that had effective growth promoting activity. In addition, the addition amount of the nitrogen source determined by the method is excessive, a large amount of unused peptides and amino acids are remained in the culture medium in the growth process, the utilization efficiency is low, the osmotic pressure of the fermentation liquor is increased, the proliferation of the strain is inhibited, and the waste of the nitrogen source is also caused.
Based on the lack of nitrogen source suitable for the efficient proliferation of bifidobacterium longum in the prior art to improve the utilization rate of the nitrogen source in the growth process of bifidobacterium longum, finding a nitrogen source suitable for the efficient proliferation of bifidobacterium longum is especially important for the commercial application of bifidobacterium longum.
Disclosure of Invention
The invention provides a method for improving the proliferation efficiency of bifidobacterium longum aiming at the problem that the proliferation of bifidobacterium longum is influenced due to low utilization rate of a nitrogen source in the culture process of the bifidobacterium longum, and the method cultures the bifidobacterium longum by using hydrophilic peptide with the proline content of 16-20% and the molecular weight of 1500-2000 Da.
In one embodiment of the invention, the hydrophilic peptide with the proline content of 16-20% and the molecular weight of 1500-2000 Da is used as the nitrogen source of the culture medium.
In one embodiment of the invention, the medium further comprises glucose, anhydrous sodium acetate, KH2PO4、K2HPO4、MgSO4·7H2O、MnSO4·4H2O、FeSO4Diammonium hydrogen citrate, tween, cysteine hydrochloride and CaCl2Yeast extract powder and carrot juice.
The invention also provides a method for improving the nitrogen source utilization rate of the bifidobacterium longum, which cultures the bifidobacterium longum by using hydrophilic peptide with the proline content of 16-20 percent and the molecular weight of 1500-2000 Da.
In one embodiment of the invention, the hydrophilic peptide with the proline content of 16-20% and the molecular weight of 1500-2000 Da is used as the nitrogen source of the culture medium.
In one embodiment of the invention, the medium further comprises glucose, anhydrous sodium acetate, KH2PO4、K2HPO4、MgSO4·7H2O、MnSO4·4H2O、FeSO4Diammonium hydrogen citrate, tween, cysteine hydrochloride and CaCl2Yeast extract powder and carrot juice.
The invention also provides a preparation method of the hydrophilic peptide with the proline content of 16-20% and the molecular weight of 1500-2000 Da, which comprises the following steps:
(1) uniformly mixing casein and water, adding protease, carrying out enzymolysis, carrying out enzyme deactivation treatment, precipitating unhydrolyzed protein, centrifuging to collect protein enzymolysis liquid, and carrying out vacuum freeze drying on the protein enzymolysis liquid to obtain casein hydrolysate dry powder;
(2) dissolving casein hydrolysate dry powder in water, performing ultrafiltration treatment by using a filter membrane with the cut-off amount of 2000Da, and collecting filtrate;
(3) concentrating and enriching the filtrate obtained in the step (2) by adopting NAK-II macroporous resin, and finally collecting components with the molecular weight of 1500-2000 Da by adopting an ultrafiltration membrane to obtain the peptide with the molecular weight of 1500-2000 Da.
In one embodiment of the invention, the concentration of the casein solution prepared in the step (1) is 65-75 g/L; carrying out enzymolysis on casein at a constant pH value, wherein the enzymolysis temperature is 45-50 ℃, the pH value is 6.5-7, and the enzymolysis time is 0.5-1 h; the protease is selected from trypsin, and the addition amount of the trypsin is 3500-4000U/g pro.
In one embodiment of the invention, the concentration of the casein solution prepared in the step (1) is 70g/L, the casein enzymolysis is carried out at a constant temperature and pH value, the temperature is 50 ℃, the pH value is 7, the enzymolysis time is 0.5h, the protease is selected from trypsin, and the addition amount is 4000U/g pro.
In one embodiment of the invention, in the step (1), the casein hydrolysate after enzymolysis is subjected to enzyme deactivation treatment by using boiling water bath for 10min, the pH is adjusted to 4.6, the protein of the hydrolysate is precipitated, and the supernatant is centrifuged at 8000r/min for 15 min.
In one embodiment of the invention, when NAK-II macroporous resin separation is performed on the casein peptide mixed component in the step (3), ethanol with the concentration of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70 and 80% (v/v) is sequentially selected as the eluent, the flow rate is 1.5mL/min, and the detection wavelength is 220 nm.
In one embodiment of the present invention, step (3) uses an ultrafiltration membrane with a cut-off of 1500Da to collect components with a molecular weight between 1500-2000 Da.
In one embodiment of the present invention, when the effect of different nitrogen sources on the growth of Bifidobacterium longum is measured, the amount of nitrogen source added is 4 g/L.
In one embodiment of the present invention, the bifidobacterium longum bacterial solution is prepared by inoculating bifidobacterium longum into a culture medium for culture, wherein the inoculation amount is 2%.
In one embodiment of the present invention, the maximum growth of Bifidobacterium longum is determined by inoculating Bifidobacterium longum into the culture medium and culturing to late logarithmic phase and determining OD600。
In one embodiment of the invention, the Bifidobacterium longum comprises Bifidobacterium longum CGMCC NO:15032, Bifidobacterium longum GDMCCNO:60461 and Bifidobacterium longum GDMCCNO: 60926.
The invention also provides a culture medium, wherein the nitrogen source in the culture medium is hydrophilic peptide with proline content of 16-20% and molecular weight of 1500-2000 Da.
In one embodiment of the invention, the medium further comprises glucose, anhydrous sodium acetate, KH2PO4、K2HPO4、MgSO4·7H2O、MnSO4·4H2O、FeSO4Diammonium hydrogen citrate, tween, cysteine hydrochloride and CaCl2Nicotinic acid, pantothenic acid, pyridoxine, riboflavin, adenine, p-aminobenzoic acid, CaCl2Yeast extract powder and carrot juice.
The invention also provides a method for improving the proliferation efficiency of the bifidobacterium longum, a method for improving the utilization rate of a nitrogen source of the bifidobacterium longum, a preparation method of hydrophilic peptide with proline content of 16-20% and molecular weight of 1500-2000 Da and application of the culture medium in culturing the bifidobacterium longum.
The invention has the beneficial effects that:
the invention provides a method for improving the proliferation efficiency of bifidobacterium longum, which is to culture the bifidobacterium longum by using a culture medium taking hydrophilic peptide with the proline content of 16-20 percent and the molecular weight of 1500-2000 Da as a nitrogen source. When the method is used for culturing Bifidobacterium longum, the Bifidobacterium longum OD is obtained at a nitrogen source culture concentration of 4g/L600Higher than 2.5, is obviously higher than other nitrogen sources and is 1.8-2.5 times of other nitrogen sources; time of generation is obvious compared with other nitrogen sourcesThe time is shortened remarkably to be less than 2.1 h.
According to the invention, peptide components are sequentially distinguished and concentrated according to the molecular weight and structural characteristics of the peptide utilized by the bifidobacterium longum and by combining a protease enzymolysis technology, ultrafiltration and a NAK-II macroporous resin separation technology, so that the peptide suitable for efficiently proliferating the bifidobacterium longum is obtained, the bifidobacterium longum is cultured by using the peptide as a culture medium of a nitrogen source, the utilization rate of the nitrogen source can reach more than 35 percent, the utilization rates of other nitrogen sources are only below 10 percent, the utilization efficiency of the nitrogen source is effectively improved, and the waste of the nitrogen source is effectively reduced.
Detailed Description
The invention is further illustrated with reference to specific examples.
Casein referred to in the following examples was purchased from Southentic Biotech, Inc.; the flavourzyme involved in the following examples was purchased from Jiangsu Bo Mei Da science and technology Co., Ltd; yeast extract FM888, beef extract, tryptone, and fish peptone, referred to in the examples below, were purchased from Angel Yeast GmbH; potassium phosphate, 2-deoxyglucose, glucose referred to in the following examples were purchased from national pharmaceutical chemicals, Inc.; bifidobacterium longum CCFM760 referred to in the following examples was purchased from the Guangdong province culture Collection of microorganisms with the accession number CGMCC NO.15032 and disclosed in the patent with the application number 201711444295.1;
bifidobacterium longum CCFM1029 is purchased from Guangdong province microorganism culture collection with the collection number GDMCC NO:60461 and is disclosed in the patent with the application number 201910137571.2;
bifidobacterium longum CCFM1109 is purchased from Guangdong province microorganism culture collection with the collection number of GDMCC NO:60926 and is disclosed in the patent with the application number of 202010161914.1.
The media involved in the following examples are as follows:
MRS-L liquid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO40.05g/L, Tween 801mL/L, agar 20g/L, cysteine hydrochloride 1 g/L.
Solid MRS-L medium: adding 1.5% agar powder based on liquid MRS-L culture medium components, and autoclaving at 115 deg.C for 20 min. Sterilizing and cooling to 50-60 ℃, adding 0.1% mupirocin into the culture medium, uniformly mixing, pouring 15mL of the culture medium into each culture dish, wrapping the culture medium by using a preservative film after the culture medium is solidified, and storing the culture medium in a refrigerator at 4 ℃ for later use.
Basic culture medium: 30g/L of anhydrous glucose, 4g/L of anhydrous sodium acetate and KH2PO4、K2HPO4Are all 2g/L, MgSO4·7H2O 0.5g/L，MnSO4·4H2O 0.005g/L，FeSO40.02g/L, 1g/L diammonium hydrogen citrate, 1mL/L Tween, 0.5g/L cysteine hydrochloride, CaCl20.2g/L carrot juice with 15% addition, 5g/L yeast extract powder, pH of 6.5, 115 deg.C, and autoclaving for 20 min.
The detection methods referred to in the following examples are as follows:
the method for measuring the nitrogen content in the fermentation liquor comprises the following steps: the method adopts a Kjeldahl method for determination, and can be specifically seen in the reference: linshuhua, anthracenone sulfate method for determining content [ J ] of crude polysaccharide in herba Peucedani, Fujian analysis and test, 2017,26(06):52-55.
Example 1: directional preparation method of peptide capable of improving multiplication efficiency of bifidobacterium longum
In this example, peptides that can be used by bifidobacterium longum were identified by experiments, and their regularity and characteristics of peptides were summarized to prepare peptides that can improve the efficiency of bifidobacterium longum proliferation. The Bifidobacterium longum selected in this embodiment is Bifidobacterium longum CGMCC NO.15032, Bifidobacterium longum GDMCC NO:60461, and Bifidobacterium longum GDMCC NO: 60926.
The specific operation is as follows:
(1) taking out the bacteria-protecting tube which is refrigerated at minus 80 ℃, dipping a small amount of bacteria liquid by using an inoculating loop after the bacteria liquid is melted, streaking the bacteria liquid on an MRS-L solid culture medium, and culturing the bacteria liquid in an anaerobic workstation at the constant temperature of 37 ℃ for 48 hours. After the bacteria grow out, selecting a single bacterial colony to inoculate in a 5mL MRS-L liquid tube, carrying out anaerobic culture for 18-24 h, and repeating the operation for 2-3 times to obtain activated bacterial liquid.
(2) Mixing casein with water at a concentration of 70g/L, adding trypsin (with an addition amount of 4000U/g pro), adjusting pH to 7, performing enzymolysis at a constant temperature of 50 ℃ and a constant pH for 0.5 hour, performing enzyme deactivation treatment in a boiling water bath for 10min after the enzymolysis is finished, adjusting pH to 4.6, precipitating unhydrolyzed casein, centrifuging at 8000r/min for 15min, taking supernatant, and collecting bacterial powder for later use after freeze-drying.
(3) And (3) carrying out ultrafiltration treatment on the trypsin hydrolysate prepared in the step (2) by adopting a membrane with the molecular weight cutoff of 2000Da to remove macromolecular proteins and polypeptides, collecting the component A with the molecular weight of less than 2000Da, and freeze-drying the component A for later use after rotary evaporation and concentration.
(4) And (3) determining the peptide segment capable of effectively transporting three strains of bifidobacterium longum in the step A by adopting a peptide transport experiment. The main operation is as follows: the medium was removed by centrifugation, and the bacterial sludge was collected and washed with 100mM potassium phosphate (containing 5mM MgSO)4) Buffer solution A of (A) cells were washed twice and resuspended to A66025. In order to cut off the power of the cells and consume the amino acids and peptides in the cells, the washed cells were cultured with 20mM 2-deoxyglucose solution at 30 ℃ for 30 min. After the power-off, the cells were washed twice with buffer A and resuspended to A660About.50, followed by dilution of the cell suspension to A with buffer A containing 0.5% glucose660And the concentration is approximately equal to 15, the mixture is pre-charged for 3min at the temperature of 30 ℃, after the pre-charging is finished, a peptide fragment with proper concentration is added to start a peptide transfer experiment, 2mL of culture medium before and after the transfer is respectively collected, and thalli are removed through a filter membrane with the diameter of 0.22 mu m. The molecular weight and the structure of the peptide segment are measured by a nano LC-MS/MS technology, and the peptide segment transported by the bifidobacterium longum is determined by comparing before and after transportation. The major characteristics of the peptide fragments transported by three strains of Bifidobacterium longum were analyzed (see Table 1 for the results of the tests). As can be seen from Table 1, the peptide fragments efficiently transported by the strain are mainly hydrophilic peptides with the molecular weight of 1500-2000 Da; the amino acid composition analysis of the transit peptide finds that the transit peptide mainly contains proline, and the calculation of the proportion of the proline in the peptide segment shows that the proline content is between 16 and 20 percent.
As a result, the peptide segment efficiently transported by the Bifidobacterium longum is mainly hydrophilic peptide with proline content of 16-20% and molecular weight of 1500-2000 Da.
TABLE 1 Bifidobacterium longum transit peptide characterization
(5) Based on the analysis, the peptide for improving the proliferation efficiency of the bifidobacterium longum is prepared, the component A collected in the step (3) is selected for subsequent preparation, and the main operations are as follows:
preparing a component A solution with the concentration of 50mg/mL, performing NAK-II macroporous resin separation on the solution A by using ethanol with the concentration of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70 and 80% (v/v), performing separation and elution according to the hydrophilicity and hydrophobicity of the component, collecting eluent, freezing, drying and collecting. Obtaining 3 components B1 (5-20%), B2 (25-50%) and B3 (55-80%), and collecting and separating the component B1.
(6) And (3) selecting an ultrafiltration membrane with the cut-off quantity of 1500Da to separate the component B1 collected in the step (5), collecting the component C (1500-2000 Da), and performing vacuum freeze drying to collect dry powder. The structure of the peptide segment in the component C is determined by adopting a liquid chromatography-mass spectrometry detection technology, and the result shows that the content of proline in the peptide segment in the component C is up to 17%.
Example 2: application of peptide suitable for high-efficiency proliferation of bifidobacterium longum
The Bifidobacterium longum selected in this embodiment is Bifidobacterium longum CGMCC NO.15032, Bifidobacterium longum GDMCC NO:60461, and Bifidobacterium longum GDMCC NO: 60926.
Three strains of bifidobacterium longum were cultured using the peptide fraction C prepared in example 1, and the highest biomass, growth generation time and nitrogen source utilization rate of the strain at that fraction were measured.
(1) Peptide component C prepared in example 1 was added to a basal medium at a concentration of 4g/L, three strains of Bifidobacterium longum were cultured, activated bacterial solutions were inoculated at an inoculum size of 2%, and OD was measured every 2 hours600And pH value, measuring the growth curve of the bifidobacterium longum under the culture condition, and measuring the highest biomass of the strain under the component; in the logarithmic phase, Bifidobacterium longum is calculated according to formula (A)Wherein G represents the growth generation of Bifidobacterium longum, A0Represents the initial absorbance of the strain at the time of entry into the logarithmic growth phase, AtThe absorbance of the fermentation broth at time t is shown.
(2) And measuring the nitrogen content before and after fermentation by a Kjeldahl method, and calculating the utilization rate of the nitrogen source before and after fermentation.
The results are shown in Table 2, and it is understood that the component C prepared in example 1 has a good proliferation effect when used to culture Bifidobacterium longum, and the final fermentation broth OD600Are all higher than 2.5, and the utilization rate of the nitrogen source is all higher than 35%, and the generation time is all less than 2.1h, thus the growth effect is good and the utilization rate of the nitrogen source is high when the component C is used for proliferation.
TABLE 2 proliferation of Bifidobacterium longum Using peptide component C prepared in example 1
Comparative example 1
The peptide component C in example 2 was replaced with yeast extract FM888, three strains of bifidobacterium longum were cultured, respectively, and the highest biomass, growth generation time, and nitrogen source utilization rate of the strain under this component were determined.
The results are shown in Table 3, and it is clear that the OD of the fermentation broth is obtained when three strains of Bifidobacterium longum yeast extract FM888 are proliferated as nitrogen source600Are all less than 1.3, the utilization rate of the nitrogen source is all less than 10%, and the generation time is all more than 2.8h, compared with the embodiment 2, the proliferation effect is poor, and the utilization rate of the nitrogen source is low.
TABLE 3 proliferation of Bifidobacterium longum by Yeast extract FM888
Comparative example 2
The peptide component C of example 2 was replaced with beef extract powder to culture three strains of bifidobacterium longum, respectively, and the highest biomass, growth generation time and nitrogen source utilization rate of the strain under the component were determined.
The results are shown in Table 4, and it can be seen that OD of the fermentation broth is obtained when three strains of Bifidobacterium longum proliferate using beef extract powder as nitrogen source600Are all less than 1.3, the utilization rate of the nitrogen source is all less than 10%, and the generation time is all more than 2.8h, compared with the embodiment 2, the proliferation effect is poor, and the utilization rate of the nitrogen source is low.
TABLE 4 proliferation of Bifidobacterium longum by beef extract
Comparative example 3
Three strains of Bifidobacterium longum were cultured with the peptide fraction C of example 2 replaced with fish peptone, respectively, and the highest biomass, growth passage time and nitrogen source utilization rate of the strain under the fraction were measured.
The results are shown in Table 5, and it is understood from the results that OD of the fermentation broth was obtained when three Bifidobacterium longum strains were proliferated using fish peptone as a nitrogen source600Are all less than 1.6, the utilization rate of the nitrogen source is all less than 10%, and the generation time is all more than 2.6h, compared with the embodiment 2, the proliferation effect is poor, and the utilization rate of the nitrogen source is low.
TABLE 5 proliferation of Bifidobacterium longum by fish peptone
Comparative example 4
Three strains of Bifidobacterium longum were cultured with the peptide fraction C of example 2 replaced with tryptone, respectively, and the highest biomass, growth passage time and nitrogen source utilization rate of the strain under the fraction were determined.
The results are shown in Table 6, and it is understood from the results that OD of the fermentation broth is obtained when three Bifidobacterium longum strains are proliferated using tryptone as a nitrogen source600Are all less than 1.6 percent of the total weight,the utilization rates of the nitrogen sources are all less than 10%, the generation time is more than 2.6h, and compared with example 2, the proliferation effect is poor, and the utilization rate of the nitrogen sources is low.
TABLE 6 proliferation of Bifidobacterium longum by tryptone
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A method for improving the proliferation efficiency of Bifidobacterium longum is characterized in that the Bifidobacterium longum is cultured by hydrophilic peptide with proline content of 16-20% and molecular weight of 1500-2000 Da.
2. The method according to claim 1, wherein the hydrophilic peptide with proline content between 16% and 20% and molecular weight between 1500Da and 2000Da is used as nitrogen source of the culture medium.
3. The method according to claim 2, wherein the medium further comprises glucose, anhydrous sodium acetate, KH2PO4、K2HPO4、MgSO4·7H2O、MnSO4·4H2O、FeSO4Diammonium hydrogen citrate, tween, cysteine hydrochloride and CaCl2Yeast extract powder and carrot juice.
4. The method according to any one of claims 1 to 3, wherein the hydrophilic peptide having a proline content of 16 to 20% and a molecular weight of 1500 to 2000Da is prepared by the following method:
(1) uniformly mixing casein and water, adding protease, carrying out enzymolysis, carrying out enzyme deactivation treatment, precipitating unhydrolyzed protein, centrifuging to collect protein enzymolysis liquid, and carrying out vacuum freeze drying on the protein enzymolysis liquid to obtain casein hydrolysate dry powder;
(2) dissolving casein hydrolysate dry powder in water, performing ultrafiltration treatment by using a filter membrane with the cut-off amount of 2000Da, and collecting filtrate;
(3) concentrating and enriching the filtrate obtained in the step (2) by adopting NAK-II macroporous resin, and finally collecting components with the molecular weight of 1500-2000 Da by adopting an ultrafiltration membrane.
5. The method according to claim 4, wherein the concentration of the casein solution prepared in the step (1) is 65-75 g/L.
6. The method according to claim 4, wherein the protease in step (1) is selected from trypsin, and the amount of trypsin is 3500-4000U/g pro.
7. The method of claim 4, wherein the step (3) uses an ultrafiltration membrane with a cut-off of 1500Da to collect the components with the molecular weight of 1500-2000 Da.
8. A culture medium is characterized in that a nitrogen source in the culture medium is hydrophilic peptide with proline content of 16-20% and molecular weight of 1500-2000 Da.
9. The culture medium according to claim 8, wherein the components of the culture medium further comprise glucose, anhydrous sodium acetate, KH2PO4、K2HPO4、MgSO4·7H2O、MnSO4·4H2O、FeSO4Diammonium hydrogen citrate, tween, cysteine hydrochloride and CaCl2Yeast extract powder and carrot juice.
10. Use of the method of any one of claims 1 to 7, or the medium of claim 8 or 9, for culturing bifidobacterium longum.
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