CN113528394A - Streptococcus thermophilus, method for preparing hyaluronic acid by using streptococcus thermophilus and application of streptococcus thermophilus - Google Patents
Streptococcus thermophilus, method for preparing hyaluronic acid by using streptococcus thermophilus and application of streptococcus thermophilus Download PDFInfo
- Publication number
- CN113528394A CN113528394A CN202110884758.6A CN202110884758A CN113528394A CN 113528394 A CN113528394 A CN 113528394A CN 202110884758 A CN202110884758 A CN 202110884758A CN 113528394 A CN113528394 A CN 113528394A
- Authority
- CN
- China
- Prior art keywords
- streptococcus thermophilus
- hyaluronic acid
- strain
- fermentation
- streptococcus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229920002674 hyaluronan Polymers 0.000 title claims abstract description 68
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 67
- 229960003160 hyaluronic acid Drugs 0.000 title claims abstract description 67
- 241000194020 Streptococcus thermophilus Species 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title abstract description 11
- 238000000855 fermentation Methods 0.000 claims abstract description 37
- 230000004151 fermentation Effects 0.000 claims abstract description 35
- 235000013305 food Nutrition 0.000 claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 239000002537 cosmetic Substances 0.000 claims description 6
- 235000020183 skimmed milk Nutrition 0.000 claims description 5
- 108010073771 Soybean Proteins Proteins 0.000 claims description 4
- 238000009629 microbiological culture Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 2
- 229940001941 soy protein Drugs 0.000 claims description 2
- 238000009776 industrial production Methods 0.000 abstract description 6
- 230000000813 microbial effect Effects 0.000 abstract description 4
- 238000012216 screening Methods 0.000 abstract description 4
- 239000002609 medium Substances 0.000 description 11
- 239000001963 growth medium Substances 0.000 description 9
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 241000194017 Streptococcus Species 0.000 description 4
- 241000194048 Streptococcus equi Species 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 235000015278 beef Nutrition 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 3
- 235000019797 dipotassium phosphate Nutrition 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 235000019341 magnesium sulphate Nutrition 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 229920000136 polysorbate Polymers 0.000 description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 description 2
- 241000192125 Firmicutes Species 0.000 description 2
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 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
- 235000010469 Glycine max Nutrition 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 108010006464 Hemolysin Proteins Proteins 0.000 description 2
- 108010003272 Hyaluronate lyase Proteins 0.000 description 2
- 102000001974 Hyaluronidases Human genes 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 2
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 241000120569 Streptococcus equi subsp. zooepidemicus Species 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000003495 flagella Anatomy 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 229940097043 glucuronic acid Drugs 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000003228 hemolysin Substances 0.000 description 2
- 229960002773 hyaluronidase Drugs 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 239000011572 manganese Substances 0.000 description 2
- 229940099596 manganese sulfate Drugs 0.000 description 2
- 235000007079 manganese sulphate Nutrition 0.000 description 2
- 239000011702 manganese sulphate Substances 0.000 description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000000630 rising effect Effects 0.000 description 2
- 239000006152 selective media Substances 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 235000019710 soybean protein Nutrition 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 2
- 239000001393 triammonium citrate Substances 0.000 description 2
- 235000011046 triammonium citrate Nutrition 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 235000013618 yogurt Nutrition 0.000 description 2
- FNEHAOQZWPHONV-UHFFFAOYSA-N 9h-carbazole;sulfuric acid Chemical compound OS(O)(=O)=O.C1=CC=C2C3=CC=CC=C3NC2=C1 FNEHAOQZWPHONV-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000193996 Streptococcus pyogenes 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
- 239000008272 agar Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004134 energy conservation Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000002095 exotoxin Substances 0.000 description 1
- 231100000776 exotoxin Toxicity 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000021474 generally recognized As safe (food) Nutrition 0.000 description 1
- 235000021473 generally recognized as safe (food ingredients) Nutrition 0.000 description 1
- 229940099552 hyaluronan Drugs 0.000 description 1
- -1 hyaluronan oligosaccharides Chemical class 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 108010009004 proteose-peptone Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000021262 sour milk Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- LZMSXDHGHZKXJD-VJANTYMQSA-N trypanothione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H]1CSSC[C@H](NC(=O)CC[C@H](N)C(O)=O)C(=O)NCC(=O)NCCCNCCCCNC(=O)CNC1=O LZMSXDHGHZKXJD-VJANTYMQSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 210000004127 vitreous body Anatomy 0.000 description 1
Images
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
-
- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of microbial fermentation engineering, and particularly discloses streptococcus thermophilus, a method for preparing hyaluronic acid by using the streptococcus thermophilus and application of the streptococcus thermophilus. The Streptococcus thermophilus (Streptococcus thermophilus) obtained by screening is a food safety strain, is safe and reliable, the yield of the high molecular weight hyaluronic acid prepared by fermenting the Streptococcus thermophilus is higher than that of the previously reported strain, the yield of the hyaluronic acid can reach 260mg/L, and large-scale culture and industrial production can be met.
Description
Technical Field
The invention relates to the technical field of microbial fermentation engineering, in particular to streptococcus thermophilus, a method for preparing hyaluronic acid by using the strain and application of the streptococcus thermophilus.
Background
Hyaluronic Acid (HA) is a linear, high molecular polysaccharide consisting of alternating N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA). HA is a major component of the extracellular matrix of skin and joints and is widely used in the medical, cosmetic and food fields.
In 1934 Meyer et al first isolated hyaluronic acid from bovine vitreous humor and subsequently in 1937 Kendall et al discovered that streptococci can produce hyaluronic acid. Hyaluronic acid is a main component of the capsule of strains such as streptococcus, pseudomonas aeruginosa and the like, and the strains capable of producing HA are mainly group A and group C of streptococcus. The group A is mainly streptococcus pyogenes and the like, but the group A is more pathogenic and is less used. Group C is less pathogenic, and most of these bacteria are used, and mainly include Streptococcus zooepidemicus (Streptococcus zooepidemicus), Streptococcus equi (Streptococcus equi), and Streptococcus equi (Streptococcus equi ssp). Most of these existing strains produce hemolysin and hyaluronidase (where hyaluronidase can break down hyaluronic acid and streptococcal hemolysin has the effects of lysing erythrocytes, killing leukocytes and poisoning the heart), the hyaluronic acid fermentation yield is low, and the hyaluronic acid produced by streptococcal fermentation cannot meet the regulatory standards due to the presence of exotoxins.
Streptococcus thermophilus is traditionally used for the preparation of dairy products, such as yoghurt and cheese. Streptococcus thermophilus is generally considered a safe organism with the ability to produce hyaluronic acid from lactose. It is reported that the streptococcus thermophilus produces hyaluronic acid in a milk-based medium with soybean peptide as a nitrogen source, the streptococcus thermophilus is used for metabolizing skimmed milk powder and various carbohydrate-supplemented synthesized hyaluronic acid in a shake flask, the maximum extracellular polysaccharide concentration is 135.8mg/L, the yield of the synthesized hyaluronic acid is low, and the large-scale culture and industrial production cannot be met.
Disclosure of Invention
The streptococcus thermophilus obtained by screening is a food-safe strain, is safe and reliable, the yield of hyaluronic acid produced by fermenting the streptococcus thermophilus is higher than that of the strain reported in the past, the yield of hyaluronic acid can reach 260mg/L, and large-scale culture and industrial production can be met.
In order to achieve the purpose, the invention adopts the technical scheme that:
in a first aspect of the invention, a strain is provided, wherein the strain has a 16s rRNA sequence shown as SEQ ID NO. 1. The strain is Streptococcus thermophilus (Streptococcus thermophilus) TSST908, and the Streptococcus thermophilus (Streptococcus thermophilus) TSST908 is deposited in China general microbiological culture collection center at 11-16 months in 2020 at the deposition address: the collection number of the microbial research institute of Chinese academy of sciences, West Lu No.1 of Chen, the rising area of Beijing, China is GDMCC No. 21193.
Streptococcus thermophilus (Streptococcus thermophilus) TSST908 of the present invention belongs to the genus Streptococcus, gram-positive bacteria. The cells are arranged in a round or oval shape and in a chain shape, have no spores and flagella, are 0.7-1.1 mu m long and 0.6-0.9 mu m wide, appear in pairs or short chains, and are optimally grown at the temperature of 38-43 ℃ and the pH value of 6.0-7.0. The Streptococcus thermophilus TSST908 has the characteristic of high-efficiency production of hyaluronic acid, the yield of the prepared hyaluronic acid can reach 260mg/L, and large-scale culture and industrial production can be met.
The Streptococcus thermophilus (Streptococcus thermophilus) TSST908 is separated from domestic Mongolian fermented yoghurt, is separated by a TPY selective medium, and is obtained by picking a single strain and expanding the single strain.
In a second aspect of the invention, the application of the strain in preparing hyaluronic acid is provided.
In a third aspect of the invention, there is provided a fermentation medium comprising the above-described strain and an acceptable carrier.
As a preferred embodiment of the fermentation medium of the present invention, the fermentation medium contains the above-mentioned strain, skim milk and soy protein.
The fermentation culture medium of the invention ensures that the hyaluronic acid prepared by fermentation has high yield and has hyaluronic acid with various molecular weights.
In a fourth aspect of the invention, there is provided the use of a fermentation medium as described above in the preparation of hyaluronic acid.
In a fifth aspect of the present invention, a method for preparing hyaluronic acid is provided, wherein hyaluronic acid is obtained by using the fermentation medium.
In some embodiments, a method of producing hyaluronic acid using streptococcus thermophilus comprises the steps of:
the screened Streptococcus thermophilus (Streptococcus thermophilus) TSST908 is placed in an MRS fermentation culture medium to be fermented for 24 hours at 42 ℃, viable bacteria of the strain are counted, the strain is transferred into the fermentation culture medium containing 10% of skimmed milk by volume fraction and 1% of soybean protein by volume fraction to be cultured by 1% of inoculation amount, the fermentation culture medium without the Streptococcus thermophilus (Streptococcus thermophilus) TSST908 is used as a control, the constant-temperature anaerobic fermentation culture is carried out for 12 hours at 42 ℃, and the HA yield and the molecular weight are detected after the fermentation is finished.
Wherein, the formula of the MRS fermentation medium is as follows: 10.0g/L of peptone, 5.0g/L of beef extract powder, 4.0g/L of yeast extract powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of triammonium citrate, 5.0g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.05g/L of manganese sulfate and 801.0 g/L of Tween, adding all the components into 1L of water, heating and dissolving, adjusting the pH value to 6.8, sterilizing at 121 ℃ for 15min, and cooling for later use.
In a sixth aspect of the present invention, there is provided a hyaluronic acid, the hyaluronic acid obtained by the above method for preparing hyaluronic acid.
In a seventh aspect of the present invention, there is provided the use of the above-mentioned strain in the fields of food, cosmetics and medical treatment.
In an eighth aspect of the present invention, there is provided the use of the above hyaluronic acid in the fields of food, cosmetics and medical treatment.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a Streptococcus thermophilus (Streptococcus thermophilus) TSST908, wherein the colony morphology is characterized in that: milky colony which is opaque and round, has a smooth surface and a convex center and a diameter of about 1.1-1.3 mm is found to be closer to a model strain in relation to the strain of the invention when compared with the model strain; the yield of the hyaluronic acid produced by fermenting the streptococcus thermophilus preserved by the method is higher than that of the strains reported in the past, the yield of the hyaluronic acid can reach 260mg/L, and the large-scale culture and industrial production can be met.
Drawings
FIG. 1 is a graph showing the change in the production of hyaluronic acid with different fermentation times using Streptococcus thermophilus TSST908 of the present invention.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
In the following examples, the composition of the TPY selective medium is: 10g/L of lactose, 5g/L of beef extract, 5g/L of yeast powder, 10g/L of casein peptone, 5g/L of soybean peptone, 2.5g/L of dipotassium phosphate, 2.5g/L of potassium dihydrogen phosphate, 0.1g/L of magnesium sulfate, 0.5g/L of Tween 800.25 g/L, L-cysteine hydrochloride and 15g/L of agar.
The MRS fermentation medium comprises the following components in percentage by weight: 10.0g/L of peptone, 5.0g/L of beef extract powder, 4.0g/L of yeast extract powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of triammonium citrate, 5.0g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.05g/L of manganese sulfate and 801.0 g/L of Tween, adding all the components into 1L of water, heating and dissolving, adjusting the pH value to 6.8, sterilizing at 121 ℃ for 15min, and cooling for later use.
Example 1 isolation, identification and cultivation of the strains
The bacterial strain is separated from natural fermented sour milk of inner Mongolia in China, is separated by a TPY selective culture medium, and is obtained by selecting a single bacterial strain and expanding the single bacterial strain.
Inoculating the screened strain into MRS liquid culture medium, culturing at 42 deg.C for 24 hr, subculturing for 3 generations, recovering strain activity, diluting the bacterial liquid to obtain 10-1～10-7Dilution gradient, by pipetting 200ul of 10 each-4、10-5、10-6、10-7Uniformly coating the gradient diluent in a MRS solid culture medium plate, performing anaerobic culture at 42 ℃ for 24h, selecting monoclonals with different shapes, sizes and colors, inoculating the monoclonals in an MRS liquid culture medium, and performing constant culture at 42 DEG CCulturing in a warm anaerobic incubator for 12h, performing gram staining and microscopic examination after the strain grows well, storing the isolate, and extracting the genomic DNA of the strain for subsequent determination and analysis.
The strain obtained by screening has the following morphological characteristics: the cells are arranged in a round or oval shape and in a chain shape, have no spores and flagella, are 0.7-1.1 mu m long and 0.6-0.9 mu m wide, appear in pairs or short chains, and are optimally grown at the temperature of 38-43 ℃ and the pH value of 6.0-7.0.
The bacterial strain obtained by screening is cultured for 24 hours at 42 ℃ on an MRS culture medium, and the morphological characteristics of the bacterial colony are as follows: milky white colony, non-transparent, round, smooth surface, convex center, diameter of about 1.1-1.3 mm. Compared with the model strain, the screened strain is found to have closer relationship with the model strain and belongs to streptococcus and gram-positive bacteria. Therefore, the strain screened by the present invention was named Streptococcus thermophilus TSST 908.
Streptococcus thermophilus (Streptococcus thermophilus) TSST908 has been deposited at the China general microbiological culture Collection center (China general microbiological culture Collection center) at 11/16/2020 at the deposition address: the collection number of the microbial research institute of Chinese academy of sciences, West Lu No.1 of Chen, the rising area of Beijing, China is GDMCC No. 21193.
Example 2 fermentation Performance verification experiment of Streptococcus thermophilus (Streptococcus thermophilus) TSST908
The screened Streptococcus thermophilus (Streptococcus thermophilus) TSST908 is fermented in MRS fermentation medium at 42 ℃ for 24h, and viable bacteria of the strain are counted. Inoculating the cells into a fermentation medium containing 10% by volume of skimmed milk and 1% by volume of soybean protein in an inoculation amount of 1%, performing anaerobic fermentation at a constant temperature of 42 ℃ for 12h by taking the fermentation medium without adding Streptococcus thermophilus (Streptococcus thermophilus) TSST908 as a control, and detecting the HA yield and molecular weight after the fermentation is finished.
1. Determination of HA production: 0.1% (w/v) Sodium Dodecyl Sulfate (SDS) was added to the fermentation broth and centrifuged at 10000 r/min for 20 min. Discarding the precipitate, adding anhydrous ethanol with twice volume into the supernatant, standing at 4 deg.C for 1h, centrifuging at 5000 r/min for 20min, resuspending the precipitate in equal volume of water, purifying for 3 times, and determining HA yield by carbazole sulfate method. WB600 was used as a control, excluding other factors. The production of Hyaluronic Acid (HA) during fermentation as a function of fermentation time is shown in FIG. 1.
As can be seen from FIG. 1, the yield of hyaluronic acid produced by fermentation of Streptococcus thermophilus TSST908 increases approximately in an S-shaped curve in 0-16 h, the yield of hyaluronic acid reaches 250g/L in 16h, the change of the yield of hyaluronic acid thereafter becomes smooth, and the highest detection value reaches about 260 g/L.
2. Method for determining molecular weight of HA: the mass average molecular weight (Mw), the number average molecular weight (Mn) and the polydispersity index of the hyaluronan oligosaccharides were determined by high performance liquid chromatography-size exclusion chromatography (HPSECMALLS-RI): ip (Ip ═ Mw/Mn). The mobile phase was 0.2mol/L, pH 7.2 phosphate buffer. Each sample was measured 3 times and the average was calculated. The molecular weight of Hyaluronic Acid (HA) as a function of fermentation time is shown in Table 1.
TABLE 1
As can be seen from Table 1, the molecular weight of hyaluronic acid produced by fermentation of Streptococcus thermophilus TSST908 slightly varies with the fermentation process, and the average value is 0.67X 106And D is about.
The Streptococcus thermophilus (Streptococcus thermophilus) TSST908 meets GRAS (American FDA evaluation of safety index of food additives), can produce intracellular polysaccharide including HA with various molecular weights, and HAs important roles in the fields of food, cosmetics and medical treatment. The existing hyaluronic acid producing strains are non-food safe strains or recombinant engineering strains, and have poor safety, and the Streptococcus thermophilus (Streptococcus thermophilus) TSST908 disclosed by the invention is a food safe Streptococcus thermophilus strain capable of naturally producing hyaluronic acid, so that the defect of industrial production of hyaluronic acid is overcome, the source of the existing hyaluronic acid producing strains is widened, and the application range of hyaluronic acid is expanded.
The Streptococcus thermophilus (Streptococcus thermophilus) TSST908 has simple culture conditions, and the hyaluronic acid produced by the strain can realize large-scale culture and meet the industrial application. The invention takes food-grade streptococcus thermophilus as a production strain, is safe and reliable, provides effective reference and reference for industrialized green production of micromolecule hyaluronic acid, and has the advantages of energy conservation, emission reduction and obvious economic benefit.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> Shandong Tian Cheng Biotechnology GmbH
<120> Streptococcus thermophilus, and method for preparing hyaluronic acid by using the strain and application thereof
<130> 2021.7.30
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 975
<212> DNA
<213> Artificial Synthesis
<400> 1
gacgtgggcg ggcgtgctat acatgcaagt agaacgctga agagaggagc ttgctcttct 60
tggatgagtt gcgaacgggt gagtaacgcg taggtaacct gccttgtagc gggggataac 120
tattggaaac gatagctaat accgcataac aatggatgac acatgtcatt tatttgaaag 180
gggcaattgc tccactacaa gatggacctg cgttgtatta gctagtaggt gaggtaatgg 240
ctcacctagg cgacgataca tagccgacct gagagggtga tcggccacac tgggactgag 300
acacggccca gactcctacg ggaggcagca gtagggaatc ttcggcaatg ggggcaaccc 360
tgaccgagca acgccgcgtg agtgaagaag gttttcggat cgtaaagctc tgttgtaagt 420
caagaacggg tgtgagagtg gaaagttcac actgtgacgg tagcttacca gaaagggacg 480
gctaactacg tgccagcagc cgcggtaata cgtaggtccc gagcgttgtc cggatttatt 540
gggcgtaaag cgagcgcagg cggtttgata agtctgaagt taaaggctgt ggctcaacca 600
tagttcgctt tggaaactgt caaacttgag tgcagaaggg gagagtggaa ttccatgtgt 660
agcggtgaaa tgcgtagata tatggaggaa caccggtggc gaaagcggct ctctggtctg 720
taactgacgc tgaggctcga aagcgtgggg agcgaacagg attagatacc ctggtagtcc 780
acgccgtaaa cgatgagtgc taggtgttgg atcctttccg ggattcagtg ccgcagctaa 840
cgcattaagc actccgcctg gggagtacga ccgcaaggtt gaactcaagg aattgacggg 900
ggcccgcaca agcggtggag catgtggttt taattcgaag caacgcgaag aacccttacc 960
agggtctttg acatc 975
Claims (10)
1. A strain, characterized in that the strain has a 16s rRNA sequence shown as SEQ ID No. 1.
2. The strain according to claim 1, wherein the strain is Streptococcus thermophilus (Streptococcus thermophilus) which has been deposited at the China general microbiological culture Collection center (CGMCC) at 11/16 of 2020 with the deposit number GDMCC No. 21193.
3. Use of a strain according to claim 1 or 2 for the preparation of hyaluronic acid.
4. A fermentation medium comprising the strain of claim 1 or 2 and an acceptable carrier.
5. The fermentation medium of claim 4, wherein the fermentation medium comprises the strain of claim 1 or 2, skim milk, and soy protein.
6. Use of a fermentation medium according to claim 4 for the preparation of hyaluronic acid.
7. A method for producing hyaluronic acid, wherein hyaluronic acid is obtained by using the fermentation medium of claim 4 or 5.
8. A hyaluronic acid obtained by the method for producing a hyaluronic acid according to claim 7.
9. Use of the strain according to claim 1 or 2 in the food, cosmetic and medical fields.
10. Use of hyaluronic acid according to claim 8 in the food, cosmetic and medical fields.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110884758.6A CN113528394A (en) | 2021-08-02 | 2021-08-02 | Streptococcus thermophilus, method for preparing hyaluronic acid by using streptococcus thermophilus and application of streptococcus thermophilus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110884758.6A CN113528394A (en) | 2021-08-02 | 2021-08-02 | Streptococcus thermophilus, method for preparing hyaluronic acid by using streptococcus thermophilus and application of streptococcus thermophilus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113528394A true CN113528394A (en) | 2021-10-22 |
Family
ID=78090196
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110884758.6A Pending CN113528394A (en) | 2021-08-02 | 2021-08-02 | Streptococcus thermophilus, method for preparing hyaluronic acid by using streptococcus thermophilus and application of streptococcus thermophilus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113528394A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI824594B (en) * | 2022-06-30 | 2023-12-01 | 捷康生技有限公司 | Novel streptococcus thermophilus strain and probiotic composition and use thereof |
| CN117338823A (en) * | 2023-12-06 | 2024-01-05 | 山东百德生物科技有限公司 | Animal ferment fermentation broth and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009112260A (en) * | 2007-11-07 | 2009-05-28 | Yakult Honsha Co Ltd | Method for producing hyaluronic acid |
| JP2012130287A (en) * | 2010-12-22 | 2012-07-12 | Yakult Honsha Co Ltd | Highly hyaluronic acid-producing lactobacillus |
| CN104164379A (en) * | 2013-04-30 | 2014-11-26 | 大江生医股份有限公司 | Probiotic bacterial strain for producing hyaluronic acid and application thereof |
-
2021
- 2021-08-02 CN CN202110884758.6A patent/CN113528394A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009112260A (en) * | 2007-11-07 | 2009-05-28 | Yakult Honsha Co Ltd | Method for producing hyaluronic acid |
| JP2012130287A (en) * | 2010-12-22 | 2012-07-12 | Yakult Honsha Co Ltd | Highly hyaluronic acid-producing lactobacillus |
| CN104164379A (en) * | 2013-04-30 | 2014-11-26 | 大江生医股份有限公司 | Probiotic bacterial strain for producing hyaluronic acid and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 新营养: "透明质酸商机，新辟益生菌蹊径", 《微信公众号》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI824594B (en) * | 2022-06-30 | 2023-12-01 | 捷康生技有限公司 | Novel streptococcus thermophilus strain and probiotic composition and use thereof |
| CN117338823A (en) * | 2023-12-06 | 2024-01-05 | 山东百德生物科技有限公司 | Animal ferment fermentation broth and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104911125B (en) | A kind of chitosan enzyme-producing bacteria and its application | |
| US20140322273A1 (en) | Strain of exopolysaccharide-secreting lactobacillus plantarum and application thereof | |
| CN113528394A (en) | Streptococcus thermophilus, method for preparing hyaluronic acid by using streptococcus thermophilus and application of streptococcus thermophilus | |
| CN107586741B (en) | Lactobacillus plantarum and application thereof in fruit enzyme products | |
| CN102994395B (en) | Aureobasidium pullulans and application thereof | |
| WO2013082915A1 (en) | Strain of exopolysaccharide-secreting lactobacillus brevis and application thereof | |
| CN107988115B (en) | Lactobacillus plantarum and composite probiotic fermentation liquor and preparation method thereof | |
| CN109880758B (en) | Lactobacillus plantarum mutant strain and mutation method and application thereof | |
| CN112574923A (en) | Streptococcus thermophilus capable of producing exopolysaccharides in high yield and application thereof | |
| CN108220201A (en) | Streptococcus thermophilus streptococcus thermophilus benshit | |
| CN105176872A (en) | Streptococcus thermophilus and application thereof | |
| CN110982759A (en) | Lactobacillus plantarum with antioxidant capacity and application thereof | |
| CN108018247B (en) | High-yield straight-chain glucan strain and glucan fermentation production method thereof | |
| CN106460021B (en) | Method for producing glucan | |
| CN110699273A (en) | Lactobacillus casei and application thereof | |
| CN110106113B (en) | Lactobacillus kefir MSR101 and application thereof | |
| CN109628340B (en) | Bacillus circulans strain for producing high-activity beta-galactosidase and breeding method thereof | |
| CN104054822A (en) | Yogurt fermentative lactic bacteria combination and fermenting agent | |
| RU2590716C1 (en) | Strain of bacteria streptococcus thermophilus strain used for preparation of cultured milk product | |
| Kabadjova et al. | Investigation of bacteriocin activity of lactic acid bacteria isolated from boza | |
| CN114292763B (en) | Saccharomyces cerevisiae for high yield of gamma-aminobutyric acid and soluble beta-glucan and application thereof | |
| CN112708577B (en) | Lactobacillus fermentum DALI02 with high intestinal adhesion and immunoregulation function and application thereof | |
| CN110484477B (en) | Lactobacillus delbrueckii subsp bulgaricus strain and application thereof | |
| CN106434786B (en) | Method for preparing exopolysaccharide by fermenting lactobacillus casei | |
| CN114181857A (en) | Antioxidant lactobacillus fermentum and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20211022 |