CN112795499A - Streptococcus thermophilus JMCC0030, and separation and purification method and application thereof - Google Patents
Streptococcus thermophilus JMCC0030, and separation and purification method and application thereof Download PDFInfo
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- CN112795499A CN112795499A CN202011344643.XA CN202011344643A CN112795499A CN 112795499 A CN112795499 A CN 112795499A CN 202011344643 A CN202011344643 A CN 202011344643A CN 112795499 A CN112795499 A CN 112795499A
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- streptococcus thermophilus
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- 241000194020 Streptococcus thermophilus Species 0.000 title claims abstract description 84
- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000000746 purification Methods 0.000 title claims abstract description 21
- 238000000926 separation method Methods 0.000 title claims abstract description 17
- 235000015140 cultured milk Nutrition 0.000 claims abstract description 43
- 230000001580 bacterial effect Effects 0.000 claims abstract description 26
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 claims abstract description 19
- 229940004208 lactobacillus bulgaricus Drugs 0.000 claims abstract description 19
- 238000004321 preservation Methods 0.000 claims abstract description 11
- 238000011160 research Methods 0.000 claims abstract description 4
- 230000002906 microbiologic effect Effects 0.000 claims abstract description 3
- 239000001963 growth medium Substances 0.000 claims description 32
- 238000000855 fermentation Methods 0.000 claims description 31
- 230000004151 fermentation Effects 0.000 claims description 31
- 244000199885 Lactobacillus bulgaricus Species 0.000 claims description 18
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 3
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 3
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- 241000186672 Lactobacillus delbrueckii subsp. bulgaricus Species 0.000 abstract 1
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- KJFMBFZCATUALV-UHFFFAOYSA-N phenolphthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2C(=O)O1 KJFMBFZCATUALV-UHFFFAOYSA-N 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 1
- 238000004945 emulsification Methods 0.000 description 1
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- FTSSQIKWUOOEGC-RULYVFMPSA-N fructooligosaccharide Chemical compound OC[C@H]1O[C@@](CO)(OC[C@@]2(OC[C@@]3(OC[C@@]4(OC[C@@]5(OC[C@@]6(OC[C@@]7(OC[C@@]8(OC[C@@]9(OC[C@@]%10(OC[C@@]%11(O[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@H]%12O)O[C@H](CO)[C@@H](O)[C@@H]%11O)O[C@H](CO)[C@@H](O)[C@@H]%10O)O[C@H](CO)[C@@H](O)[C@@H]9O)O[C@H](CO)[C@@H](O)[C@@H]8O)O[C@H](CO)[C@@H](O)[C@@H]7O)O[C@H](CO)[C@@H](O)[C@@H]6O)O[C@H](CO)[C@@H](O)[C@@H]5O)O[C@H](CO)[C@@H](O)[C@@H]4O)O[C@H](CO)[C@@H](O)[C@@H]3O)O[C@H](CO)[C@@H](O)[C@@H]2O)[C@@H](O)[C@@H]1O FTSSQIKWUOOEGC-RULYVFMPSA-N 0.000 description 1
- 229940107187 fructooligosaccharide Drugs 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1238—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt using specific L. bulgaricus or S. thermophilus microorganisms; using entrapped or encapsulated yoghurt bacteria; Physical or chemical treatment of L. bulgaricus or S. thermophilus cultures; Fermentation only with L. bulgaricus or only with S. thermophilus
-
- 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
- C12N1/02—Separating microorganisms from their culture media
-
- 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
- A23V2400/123—Bulgaricus
-
- 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
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/249—Thermophilus
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of bioengineering, and discloses streptococcus thermophilus JMCC0030, a separation and purification method thereof and application thereof. The bacterial strain is screened from Xinjiang traditional fermented products after collection, separation and purification, and is currently preserved in the bacterial preservation center of the institute of microbiological research of the Chinese academy of sciences with the preservation number of CGMCC NO. 19075. After being compounded with the Lactobacillus bulgaricus JMCC0018, the Streptococcus thermophilus JMCC0030 provided by the invention is used for preparing fermented milk, and the obtained fermented milk has good fragrance and viscosity and is capable of obviously improving osteoporosis. The invention is suitable for separation, purification and application of bacterial strains, in particular to separation, purification and application of streptococcus thermophilus JMCC 0030.
Description
Technical Field
The invention belongs to the field of bioengineering, and relates to a strain, a screening method and application, in particular to streptococcus thermophilus JMCC0030, and the screening method and application thereof.
Background
Streptococcus thermophilus is one of the most widely applied fermentation strains in the current industrial production, and the development and the utilization of the Streptococcus thermophilus in dairy products are highly regarded by the dairy industry at home and abroad. Streptococcus thermophilus, which is a key strain in the production of the food industry, is considered as a Generally Recognized As Safe (GRAS) component, not only can be used for preparing a plurality of fermented dairy products (such as yoghourt and cheese), but also can be used for preparing functional products as potential beneficial bacteria through the functional activity (such as exopolysaccharide, bacteriocin and the like) of the Streptococcus thermophilus. However, the leavening agents and strains of dairy enterprises in China still present import-dependent situations, and the strain development technology has certain gap with foreign dairy enterprises. Therefore, screening, separating and obtaining excellent zymocyte strains with independent intellectual property rights become a problem to be solved urgently by the domestic dairy enterprises.
Taiwan patent application with application number of 106120675 discloses an application of Streptococcus thermophilus TCI633 strain and metabolite thereof in preventing and/or treating osteoporosis. The streptococcus thermophilus TCI1633 and the food containing the strain are disclosed, wherein the aim of preventing or treating osteoporosis is achieved by inhibiting the activity and differentiation of osteoclasts. However, factors causing osteoporosis also include hormone imbalance in vivo, which results in slow absorption of calcium and other nutrient elements, so that nutrient substances lost in the metabolic process cannot be timely supplemented, and then osteoporosis is caused. Meanwhile, there is no directionality in inhibiting the differentiation of cells into osteocytes, and it is also possible that the inhibition of cell differentiation into other beneficial cells may cause other symptoms.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide streptococcus thermophilus JMCC0030, a separation and purification method thereof and application thereof, so as to improve the hormone level in the body and promote the absorption of nutrient substances, thereby improving bones
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the Streptococcus thermophilus JMCC0030 is deposited in the culture collection center of the institute of microbiology of academy of sciences of China with the preservation number of CGMCC NO.19075 and the Latin name is Streptococcus thermophilus.
As a limitation of the invention, the streptococcus thermophilus JMCC0030 is separated and screened from Xinjiang traditional fermentation samples;
as another limitation of the invention, the 16SrDNA sequence of the Streptococcus thermophilus JMCC0030 is as follows:
AAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGG GAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTA CAATCCGAACTGAGATTGGCTTTAAGAGATTAGCTCGCCGTCACCGACTCGCAACTCGTTGTACCAACCATTG TAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATTAC CGGCAGTCTCGCTAGAGTGCCCAACTGAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAA CCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGATGTACCGAAGTAACTTTCTAT CTCTAGAAATAGCATCGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCC ACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTA ATGCGTTAGCTGCGGCACTGAATCCCGGAAAGGATCCAACACCTAGCACTCATCGTTTACGGCGTGGACTACC AGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGAGAGCCGCTTTCG CCACCGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCCCTTCTGCACTCA AGTTTGACAGTTTCCAAAGCGAACTATGGTTGAGCCACAGCCTTTAACTTCAGACTTATCAAACCGCCTGCGC TCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGC CGTCCCTTTCTGGTAAGCTACCGTCACAGTGTGAACTTTCCACTCTCACACCCGTTCTTGACTTACAACAGAG CTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCGGTCAGGGTTGCCCCCATTGCCGAAGATTCC CTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCT ATGTATCGTCGCCTAGGTGAGCCATTACCTCACCTACTAGCTAATACAACGCAGGTCCATCTTGTAGTGGAAC AATTGCCCCTTTCAAATAAATGACATGTGTCATCCATTGTTATGCGGTATTAGCTATCGTTTCCAATAGTTAT CCCCCGCTACAAGGCAGGTTACCTACGCGTTACTCACCCGTTCGCAACTCATCCAAGAAGAGCAAGCTCCTCT CTTCAGCGTTCTACT；
as a second limitation of the invention, the sequence of the Phos gene of the Streptococcus thermophilus JMCC0030 is as follows:
GAACCCGGATACCTGACTCTCAGCACTTGTGGGTGAACCATACCAGCACCAAGGATCTCAATCCAACCT GTATTCTTGCATACGTTACATCCTTTACCACCACACTTGAAGCATGACACGTCAACCTCAACGGAAGGTTCAG TGAATGGGAAGTAAGAAGGACGCAAACGGATTCGACGTTCTGCACCAAACATTTTTTGAATAATCATCTCAAG CGTTCCCTTCAGATCACCCATTGAGATGTTTTTACCAACGACCAAACCTTCGATTTGGTGAAACTGGTGGCTG TGAGTCGCATCATCGGTATCACGACGGAAAACACGTCCTGGTGAGATCATCTTAAGAGGACCTTTAGAAAAAT CATGTTTATCAAGTGTACGAGCTTGGACAGGACTTGTATGAGTGCGAAGCAAGATTTCTTCGGTAATGTAGAA AGTATCTTGCATATCACGAGGCTGGATGGGTCT；
the invention also provides a separation and purification method of streptococcus thermophilus JMCC0030, which is carried out according to the following steps:
collecting samples
Adding a Xinjiang traditional fermentation sample into physiological saline, and fully and uniformly mixing to obtain a sample A;
wherein the volume ratio of the fermentation sample to the normal saline is 1: 9;
② sample enrichment
Adding the sample A into an MRS liquid culture medium, and culturing at 35-40 ℃ for 62-82 h to obtain a culture solution B;
wherein the volume ratio of the sample A to the MRS liquid culture medium is 1: 10-100;
③ separating and screening strains
Taking the culture solution B, and performing 10-fold gradient dilution with sterile physiological saline with the concentration of 0.9%, wherein the gradient dilution is 10 times-1、10-2、10-3、10-4、10-5Multiplying to obtain bacterial suspension C1-C5 in turn;
taking an MRS solid culture medium, melting, sequentially pouring the MRS solid culture medium into five sterilized culture dishes, cooling and completely solidifying to obtain culture media D1-D5, respectively sucking 0.1mL of bacterial suspensions C1-C5 with various concentration gradients, coating the bacterial suspensions on the culture media D1-D5 in a one-to-one correspondence manner, inverting a flat plate, placing the flat plate in an anaerobic culture environment at 35-40 ℃ for 62-82 h, and observing the growth condition of bacterial colonies;
after the plate has the typical bacteria, selecting a corresponding single bacterial colony E according to the bacterial colony characteristics of the standard streptococcus thermophilus and reference related literature pictures;
purification of bacterial strain
Selecting a selected single colony E, streaking and inoculating a colony culture to an MRS solid culture medium for primary culture, and culturing in an aerobic environment at the temperature of 35-40 ℃ for 62-82 hours to obtain a single colony F;
continuously streaking and inoculating the single colony F to an MRS solid culture medium for secondary culture, and carrying out aerobic environment culture at 35-40 ℃ for 62-82 h to obtain a single colony G;
then, continuously carrying out streak inoculation on the single colony G to an MRS solid culture medium for third culture, and carrying out aerobic environment culture at 35-40 ℃ for 62-82H to obtain a pure culture H, namely the streptococcus thermophilus JMCC0030 strain;
fifthly, preservation of the strain
Mixing pure culture H and sterile glycerol with the mass fraction of 50% according to the proportion of 1:1 proportion, placing the mixture into a strain storage tube, uniformly mixing the mixture, storing the mixture at a temperature of between 80 ℃ below zero and 70 ℃ below zero, and simultaneously inoculating an MRS solid culture medium test tube inclined plane for temporary storage.
As a limitation of the above separation and purification method, the MRS liquid medium, in parts by weight, is prepared from the following raw materials as active ingredients: 10 parts of casein peptone, 10 parts of beef extract, 5 parts of yeast extract, 20 parts of glucose, 5 parts of sodium acetate, 2 parts of citric acid diamine, 801 parts of tween-K2HPO42 parts of MgSO 24·7H20.2 part of O and MnSO4·7H20.05 part of O and 1000 parts of distilled water;
as a further limitation of the above separation and purification method, the MRS solid medium is obtained by adding 15 parts of agar to 1000 parts of the MRS liquid medium;
the invention also provides an application of streptococcus thermophilus JMCC0030, and the strain is used for preparing fermented milk after being compounded with lactobacillus bulgaricus JMCC 0018;
wherein the Lactobacillus bulgaricus JMCC0018 is preserved in the culture collection center of the institute of microbiological research of the academy of sciences of China with the preservation number of CGMCC NO. 14425.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the following beneficial effects:
(1) the streptococcus thermophilus JMCC0030 provided by the invention is compounded with the high-yield acetaldehyde lactobacillus bulgaricus JMCC0018 disclosed in the prior application document 201711359762.0 to prepare fermented milk, so that the acetaldehyde yield is increased, the flavor enhancement function of JMCC0018 is kept, the viscosity of the fermented milk is increased, and the prepared fermented milk has good fragrance and high viscosity;
(2) the streptococcus thermophilus JMCC0030 provided by the invention can produce high-concentration beneficial active substances, namely short-chain fatty acids (SCFAs, which refer to organic fatty acids with the carbon number less than 6, and mainly comprise formic acid, acetic acid, propionic acid, butyric acid and the like) in the fermentation process. The functions of the SCFAs are not limited to acidification of intestinal tracts, but also participate in gastrointestinal physiology, immune function and host metabolism, even participate in development and homeostasis of a central nervous system, and play an important role in body health;
(3) after being taken into the body, the streptococcus thermophilus JMCC0030 provided by the invention can treat estradiol (E) in the body2) Intervention is carried out, so that the level of E2 is increased, and the increase of E2 can promote intestinal calcium absorption and reduce urinary calcium excretion, thereby improving bone density;
in conclusion, after the streptococcus thermophilus JMCC0030 and the Lactobacillus bulgaricus JMCC0018 are compounded, fermented milk prepared by fermentation has good fragrance and high viscosity, and can improve osteoporosis.
The invention is suitable for separation, purification and application of bacterial strains, in particular to separation, purification and application of streptococcus thermophilus JMCC 0030.
Detailed Description
The following description is given in conjunction with preferred embodiments of the present invention. It should be understood that the description of the preferred embodiment is only for purposes of illustration and understanding, and is not intended to limit the invention.
Example 1 Streptococcus thermophilus JMCC0030
The embodiment is streptococcus thermophilus JMCC0030 which is separated and screened from Xinjiang traditional fermentation products and is stored in the culture collection center of the institute of microbiology of Chinese academy of sciences, wherein the storage address is No. 3 of Xilu No.1 of Beijing Korean district, and the preservation number is CGMCC NO. 19075.
The 16SrDNA sequence is as follows:
AAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAAC GTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAAT CCGAACTGAGATTGGCTTTAAGAGATTAGCTCGCCGTCACCGACTCGCAACTCGTTGTACCAACCATTGTAGC ACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATTACCGGC AGTCTCGCTAGAGTGCCCAACTGAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCA ACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGATGTACCGAAGTAACTTTCTATCTCT AGAAATAGCATCGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCG CTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGC GTTAGCTGCGGCACTGAATCCCGGAAAGGATCCAACACCTAGCACTCATCGTTTACGGCGTGGACTACCAGGG TATCTAATCCTGTTCGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGAGAGCCGCTTTCGCCAC CGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCCCTTCTGCACTCAAGTT TGACAGTTTCCAAAGCGAACTATGGTTGAGCCACAGCCTTTAACTTCAGACTTATCAAACCGCCTGCGCTCGC TTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTC CCTTTCTGGTAAGCTACCGTCACAGTGTGAACTTTCCACTCTCACACCCGTTCTTGACTTACAACAGAGCTTT ACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCGGTCAGGGTTGCCCCCATTGCCGAAGATTCCCTAC TGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTATGT ATCGTCGCCTAGGTGAGCCATTACCTCACCTACTAGCTAATACAACGCAGGTCCATCTTGTAGTGGAACAATT GCCCCTTTCAAATAAATGACATGTGTCATCCATTGTTATGCGGTATTAGCTATCGTTTCCAATAGTTATCCCC CGCTACAAGGCAGGTTACCTACGCGTTACTCACCCGTTCGCAACTCATCCAAGAAGAGCAAGCTCCTCTCTTC AGCGTTCTACT。
the Phos gene sequence is as follows:
GAACCCGGATACCTGACTCTCAGCACTTGTGGGTGAACCATACCAGCACCAAGGATCTCAATCCAACCTGTAT TCTTGCATACGTTACATCCTTTACCACCACACTTGAAGCATGACACGTCAACCTCAACGGAAGGTTCAGTGAA TGGGAAGTAAGAAGGACGCAAACGGATTCGACGTTCTGCACCAAACATTTTTTGAATAATCATCTCAAGCGTT CCCTTCAGATCACCCATTGAGATGTTTTTACCAACGACCAAACCTTCGATTTGGTGAAACTGGTGGCTGTGAG TCGCATCATCGGTATCACGACGGAAAACACGTCCTGGTGAGATCATCTTAAGAGGACCTTTAGAAAAATCATG TTTATCAAGTGTACGAGCTTGGACAGGACTTGTATGAGTGCGAAGCAAGATTTCTTCGGTAATGTAGAAAGTA TCTTGCATATCACGAGGCTGGATGGGTCT。
example 2 method for separating and purifying Streptococcus thermophilus JMCC0030
This example is a method for separating and purifying streptococcus thermophilus JMCC0030, which comprises the following steps:
(ii) sample Collection
Adding 25mL of Xinjiang traditional fermented product (traditional fermented dairy product prepared by herdsman in Xinjiang Kashi area) into 225mL of physiological saline, and mixing uniformly to obtain a sample A;
② sample enrichment
Taking 2mL of sample A, adding the sample A into 100mLMRS liquid culture medium, and culturing for 72h at 37 ℃ to obtain culture solution B;
③ separating and screening strains
Taking 1mL of culture solution B, diluting with sterile physiological saline with concentration of 0.9% by gradient of 10 times, respectively diluting with gradient of 10-1、10-2、10-3、10-4、10-5Doubling to obtain bacterial suspension C1-C5;
taking an MRS solid culture medium, melting, pouring the MRS solid culture medium into a culture dish, cooling and completely solidifying to obtain culture media D1-D5, respectively sucking 0.1mL of each of bacterial suspensions C1-C5 with each concentration gradient, coating the bacterial suspensions C1-C5 on different culture media D1-D5, inverting the flat plate, placing the flat plate in an anaerobic culture environment at 37 ℃ for 72 hours, and observing the growth condition of bacterial colonies;
after the plate has the typical bacteria, selecting a corresponding single bacterial colony E according to the bacterial colony characteristics of the standard streptococcus thermophilus;
purification of bacterial strain
Selecting a selected single colony E, streaking and inoculating a colony culture to an MRS solid culture medium for primary culture under the condition of aerobic environment culture at 37 ℃ for 72 hours to obtain a single colony F;
then, continuously streaking and inoculating the single colony F to an MRS solid culture medium for secondary culture under the condition of aerobic environment culture at 37 ℃ for 72 hours to obtain a single colony G;
then, continuously streaking and inoculating the single colony G to an MRS solid culture medium for third culture under the condition of aerobic environment culture at 37 ℃ for 72 hours to obtain a pure culture H;
fifthly, preservation of the strain
Respectively taking 800 mu L of pure culture H and sterile glycerol with the mass fraction of 50%, placing the pure culture H and the sterile glycerol into a strain storage tube, uniformly mixing, storing at-70 ℃, and simultaneously inoculating an MRS solid culture medium test tube inclined plane for temporary storage;
in this example, the MRS liquid medium is prepared from 10g casein peptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate, 2g diamine citrate, 1g tween-80, and 2g K2HPO4、0.2gMgSO4·7H2O、 0.05gMnSO4·7H2O and 1000mL of distilled water are fully dissolved to obtain the product;
wherein, the MRS solid culture medium is prepared by adding 15g of agar into 1000mLMRS liquid culture medium, melting, mixing uniformly and solidifying.
Example 3-6 method for separating and purifying Streptococcus thermophilus JMCC0030
Embodiments 3 to 6 provide a method for separating and purifying streptococcus thermophilus JMCC0030, which is substantially the same as the method of embodiment 2, except that technical parameters of the separation and purification process are different, and specific parameters are shown in table 1:
TABLE 1 examples 3-6 isolation and purification procedures and parameters
The other parameters were the same as in example 2.
Example 7 bacteriological basic characteristics of Streptococcus thermophilus JMCC0030
This example is the basic bacteriological characteristics of streptococcus thermophilus JMCC0030, as shown in table 2:
TABLE 2 basic characteristics of Streptococcus thermophilus JMCC0030
| Experimental project | Results | Experimental project | Results |
| Gram stain | Positive for | Cell morphology | Spherical shape |
| Oxidase enzyme | - | Contact enzyme | - |
Example 8 sugar fermentation Properties of Streptococcus thermophilus JMCC0030
This example shows the sugar fermentation properties of Streptococcus thermophilus JMCC 0030. The experimental method for the sugar fermentation characteristics comprises the following steps: selecting a single colony of streptococcus thermophilus JMCC0030, inoculating the single colony into a sterilized liquid MRS culture medium, culturing for 24h at 37 ℃, respectively inoculating the obtained bacterial suspension into different sugar fermentation tubes (the content of the specific sugar fermentation tube is shown in table 3), culturing for 48h at 37 ℃, and observing color change. The results of the identification of the sugar fermentation characteristics are shown in Table 3:
TABLE 3 identification of fermentation characteristics of Streptococcus thermophilus JMCC0030 sugars
Note: "+" indicates fermentation utilization; "-" indicates no fermentative utilization.
Example 9 molecular biological identification of Streptococcus thermophilus JMCC0030
The streptococcus thermophilus JMCC0030 is taken for molecular biological identification, and finally determined as the streptococcus thermophilus by DNA extraction, PCR amplification and 16SrDNA sequencing on NCBI website blast.
The 16SrDNA sequencing result is as follows:
AAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGG GAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTA CAATCCGAACTGAGATTGGCTTTAAGAGATTAGCTCGCCGTCACCGACTCGCAACTCGTTGTACCAACCATTG TAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATTAC CGGCAGTCTCGCTAGAGTGCCCAACTGAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAA CCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGATGTACCGAAGTAACTTTCTAT CTCTAGAAATAGCATCGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCC ACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTA ATGCGTTAGCTGCGGCACTGAATCCCGGAAAGGATCCAACACCTAGCACTCATCGTTTACGGCGTGGACTACC AGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGAGAGCCGCTTTCG CCACCGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCCCTTCTGCACTCA AGTTTGACAGTTTCCAAAGCGAACTATGGTTGAGCCACAGCCTTTAACTTCAGACTTATCAAACCGCCTGCGC TCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGC CGTCCCTTTCTGGTAAGCTACCGTCACAGTGTGAACTTTCCACTCTCACACCCGTTCTTGACTTACAACAGAG CTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCGGTCAGGGTTGCCCCCATTGCCGAAGATTCC CTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCT ATGTATCGTCGCCTAGGTGAGCCATTACCTCACCTACTAGCTAATACAACGCAGGTCCATCTTGTAGTGGAAC AATTGCCCCTTTCAAATAAATGACATGTGTCATCCATTGTTATGCGGTATTAGCTATCGTTTCCAATAGTTAT CCCCCGCTACAAGGCAGGTTACCTACGCGTTACTCACCCGTTCGCAACTCATCCAAGAAGAGCAAGCTCCTCT CTTCAGCGTTCTACT。
the Phos gene sequence is as follows:
GAACCCGGATACCTGACTCTCAGCACTTGTGGGTGAACCATACCAGCACCAAGGATCTCAATCCAACCTGTAT TCTTGCATACGTTACATCCTTTACCACCACACTTGAAGCATGACACGTCAACCTCAACGGAAGGTTCAGTGAA TGGGAAGTAAGAAGGACGCAAACGGATTCGACGTTCTGCACCAAACATTTTTTGAATAATCATCTCAAGCGTT CCCTTCAGATCACCCATTGAGATGTTTTTACCAACGACCAAACCTTCGATTTGGTGAAACTGGTGGCTGTGAG TCGCATCATCGGTATCACGACGGAAAACACGTCCTGGTGAGATCATCTTAAGAGGACCTTTAGAAAAATCATG TTTATCAAGTGTACGAGCTTGGACAGGACTTGTATGAGTGCGAAGCAAGATTTCTTCGGTAATGTAGAAAGTA TCTTGCATATCACGAGGCTGGATGGGTCT。
example 10 comparative experiment on fermentation characteristics of Streptococcus thermophilus JMCC0030
The invention provides a leavening agent compounded by lactobacillus bulgaricus JMCC0018 and streptococcus thermophilus JMCC0030 based on the fermentation characteristic research of lactobacillus bulgaricus JMCC0018 strain for high yield of acetaldehyde in the early stage, and the leavening agent is utilized to ferment to prepare dairy products, and the specific fermentation process is carried out according to the following steps:
first, strain activation
Lactobacillus bulgaricus JMCC0018 and Streptococcus thermophilus JMCC0030 are respectively activated according to the following steps:
1) taking 10g casein peptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate, 2g diamine citrate, 1g tween-80 and K2HPO4：2g、0.2g MgSO4·7H2O、0.05g MnSO4·7H2And fully dissolving and uniformly mixing O, 15g of agar and 1000mL of distilled water, and adjusting the pH value to 6.8 to obtain the liquid MRS culture medium.
Inoculating lactobacillus bulgaricus JMCC0018 and streptococcus thermophilus JMCC0030 to a liquid MRS culture medium respectively (inoculating lactobacillus bulgaricus JMCC0018 to one liquid MRS culture medium and inoculating streptococcus thermophilus JMCC0030 to the other liquid MRS culture medium), and culturing at 37 ℃ for 48h to obtain a corresponding first generation;
2) adding 1mL of corresponding first generation into 9mL of skimmed milk, and culturing for 16h to serve as corresponding second generation;
3) adding 1mL of the corresponding second generation into 9mL of skimmed milk, and culturing for 16h to obtain a third generation of activated strain;
second, preparation of fungus powder
The lactobacillus bulgaricus JMCC0018 and streptococcus thermophilus JMCC0030 bacterial powder are prepared according to the following steps:
1) putting the corresponding activated strain into a fermentation tank at 37 ℃ for fermentation for 10 hours;
2) after the fermentation liquor is cooled for 1h at 4 ℃, separating thalli at low temperature (4 ℃) by a tubular centrifuge, and adding a freeze-drying protective agent for emulsification to obtain corresponding emulsion;
3) freeze-drying the corresponding emulsion in a vacuum freeze-dryer at-40 deg.C, detecting water activity with a metal probe until the water activity is less than 0.2 (conventional method), to obtain corresponding freeze-dried bacteria;
4) pulverizing the corresponding lyophilized bacteria in a pulverizer, sieving with 40 mesh sieve to obtain corresponding single strain powder with viable count not less than 2 × 1010cfu/g；
Three, compound
Respectively taking lactobacillus bulgaricus JMCC0018 bacterial powder and streptococcus thermophilus JMCC0030 bacterial powder, and mixing the two components according to the viable bacteria number ratio of 1: 1000 to obtain the leaven.
Fourthly, fermentation
Taking 80kg of fresh milk, 10kg of white granulated sugar and 10kg of fructo-oligosaccharide, uniformly blending, homogenizing at 60 ℃, 15MPa, sterilizing at 95 ℃ for 300s, cooling to 37 ℃, adding 10g of leavening agent, fermenting at 37 ℃ until the pH value is 4.2, stopping fermentation, uniformly stirring to obtain the fermented milk I, and performing sensory evaluation on a fermented sample to obtain smooth fermented milk with moderate viscosity and acidity and abundant fragrance. The streptococcus thermophilus JMCC0030 has excellent fermentation characteristics, and can be compounded with lactobacillus bulgaricus JMCC0018 with high acetic acid yield to prepare a fermentation agent for preparing a fermented dairy product.
Fermented milk II and fermented milk III were prepared according to the third fermentation step described above using Lactobacillus bulgaricus JMCC0018 powder and Streptococcus thermophilus JMCC0030 powder, respectively, and the comparison results are shown in Table 4.
Table 4: comparison table for fermentation characteristics of strain
| Taste of the product | Viscosity of the oil | Acidity of the solution | Fragrance | |
| Fermented milk I | Smooth and slippery | Is moderate | Is moderate | Rich in flavor |
| Fermented milk II | Light astringent | Is poor | Is weaker | In general |
| Fermented milk III | Dryness and astringency | Viscosity of viscous material | Is strong and strong | Rarefaction |
Example 11 determination of acetaldehyde in fermented milk with Streptococcus thermophilus JMCC0030 Strain
The acetaldehyde contents generated during storage of the fermented milks I and II of example 10 were measured every 7 days by using a liquid chromatograph, and the results are shown in Table 5 (unit: μ g/mL), and the data were statistically analyzed by GraphPad Prism 5 software, and the data were expressed as mean. + -. standard deviation, and the data were analyzed by one-way variance T-test for comparison between two groups, and P <0.05 was found to have a very significant difference, as follows.
TABLE 5 change in acetaldehyde amount during preservation of fermented milk (unit: μ g/ml)
| Storage time | 0d | 7d | 14d | 21d |
| Fermented milk I | 0.19±0.06 | 10.92±0.17 | 23.79±0.24a | 25.56±0.33a |
| Fermented milk II | 0.22±0.09 | 9.78±0.21 | 20.52±0.19 | 21.34±0.27 |
Note: a, p <0.05 compared to fermented milk II; d, fermentation days.
The experimental result shows that the acetaldehyde content of the fermented milk I is obviously higher than that of the fermented milk II from the 14 th day of preservation, and the acetaldehyde generation condition of the fermented milk obtained by the fermentation after the streptococcus thermophilus JMCC0030 and the lactobacillus bulgaricus JMCC0018 are compounded is obviously improved, namely the acetaldehyde content of the fermented product can be improved by preparing the fermenting agent from the streptococcus thermophilus JMCC0030 on the basis of the lactobacillus bulgaricus JMCC0018 strain added with high-yield acetaldehyde.
Example 12 measurement of viscosity of fermented milk with Streptococcus thermophilus JMCC0030 Strain Complex
The fermented milks I and II of example 10 were each tested every 7 days with a DV2TLVTJ0 type viscometer at 25 ℃ and 60r/min for 1min, and the results are shown in Table 6 (unit: cp).
TABLE 6 change in viscosity (unit: cp) during preservation of fermented milk
| Storage time | 0d | 7d | 14d | 21d |
| Fermented milk I | 1029±4 | 1084±2a | 1149±6a | 1237±2a |
| Fermented milk II | 1026±3 | 1039±7 | 1058±4 | 1083±4 |
Note: a, p <0.05 compared to fermented milk II; d, fermentation days.
The experimental results show that from day 7 onwards, the viscosity of fermented milk I is significantly higher than that of fermented milk II and persists until day 21, indicating that the addition of streptococcus thermophilus JMCC0030 significantly increases the viscosity of the fermented milk.
Examples 11 to 12 show that after the streptococcus thermophilus JMCC0030 provided by the invention is compounded with the previously identified lactobacillus bulgaricus JMCCOO18 with high acetaldehyde yield, the acetaldehyde content of the fermented milk is not obviously changed, but the viscosity of the fermented milk is obviously improved, so that the taste and texture of the fermented milk are improved.
Example 13 measurement of SCFAs content in Streptococcus thermophilus JMCC0030 Strain Complex fermented milk
The method for analyzing the content of SCFAs in the streptococcus thermophilus JMCC0030 strain compound fermented milk by using a gas chromatography method comprises the following specific steps:
adjusting chromatographic conditions
The injection port temperature was set at 270 deg.C, the detector temperature at 280 deg.C, the injection amount at 1 μ L, and the carrier gas flow rate at 1 mL/min. Setting a temperature program: 50 ℃ for 10min, then raising the temperature to 230 ℃ for 9 min.
② pretreatment of samples and standards
And respectively taking 10mL of the fermented milk I and 10mL of the fermented milk II, respectively placing the fermented milk I and the fermented milk II in 15mL of centrifuge tubes, centrifuging, respectively adding 2 drops of phenolphthalein reagent into 4mL of obtained supernatant in a new centrifuge tube, adjusting the liquid color to light pink by using KOH, uniformly mixing and centrifuging after the volume of triple-distilled water is adjusted to 5mL, and thus obtaining the corresponding sample X and sample Y.
Taking formic acid, acetic acid, propionic acid and butyric acid standard substances according to the proportion of 1: dissolving at 400 mass ratio, and adjusting the standard solution to light pink according to the steps. The obtained solution was subjected to gradient dilution of 7 grades of mass concentration to achieve mass concentrations of 2000mg/L, 1000mg/L, 500mg/L, 200mg/L, 100mg/L, 50mg/L, and 20mg/L, respectively.
③ derivatization
And (3) putting X, Y samples and 0.5mL of all standard samples into a sample feeding bottle, respectively and sequentially adding 2mL of phosphate buffer solution, 2mL of derivatization reagent and 1mL of acetone, carrying out water bath at 100 ℃ for 40min, and cooling to room temperature to obtain corresponding derivatized samples and standard samples. According to the following steps: 1 volume of the derivatized sample was extracted with n-hexane, and the obtained extract was analyzed by gas chromatography, the analysis results of which are shown in Table 7.
TABLE 7 recovery and background values for acidified milk
The adding standard quantity represents the concentration of the added standard substance, the background value represents the measured value of the sample, the adding standard result represents the measured value of the adding standard sample, and the adding standard recovery rate is calculated by the following method:
the standard recovery rate is (standard result mean-background value)/standard quantity is multiplied by 100%
Analysis table 7 shows that the concentrations of formic acid, acetic acid, propionic acid and butyric acid in sample X are higher than in sample Y, indicating that the content of SCFAs in fermented milk I is higher.
Example 14 Streptococcus thermophilus JMCC0030 Strain ameliorates osteoporosis Properties
The streptococcus thermophilus strain JMCC0030 provided in example 1 was obtained and used in the SD rat ovarian removal osteoporosis test to obtain the osteoporosis improving properties of the strain. The specific test steps are as follows:
30 clean female SD rats with the body mass of 120g +/-10 g are purchased from the experimental animal center of Hebei medical university, and the production license number is as follows: SCXK (Ji) 2013-1003. All rats are raised in cages in the same room, 5 rats are raised in each cage, the rats are fed by common basic feed, water freely enters the cages, and the day and night illumination change period is 12 hours, and the rats are dark within 12 hours. The environmental temperature is controlled to be 23 +/-2 ℃ and the relative humidity is controlled to be 55-65 percent.
30 rats were randomly divided into 3 groups: blank control group, model group, JMCC0030 intervention group. Except for the placebo group, two groups of rats were ovariectomized to establish an estrogen-deficient osteoporosis model. After the modeling is successful, carrying out oral gavage treatment on rats of each group, wherein the blank control group and the model group are subjected to gavage distilled water according to 1mL/100 g.BW once a day for 6 weeks; the JMCC0030 intervention group is filled with JMCC0030 suspension (the viable count is 10) according to 1mL/100 g.BW6cfu/mL), fresh daily for 6 weeks.
After all rats are fasted for 12 hours without water supply after intervention is finished, blood is taken from eyeballs and centrifuged, and the obtained serum is analyzed for estradiol (E) in the serum of the rats by an ELISA method2) The contents and specific data are shown in Table 8. The left femur was taken after the treatment of the rats and subjected to bone density measurement, the measurement results of which are shown in Table 9.
TABLE 8 Streptococcus thermophilus JMCC0030 treatment of E in rat serum2Influence of (2)
| Group of | E2(pg/mL) |
| Blank control group | 237.39±5.82 |
| Model set | 179.13±9.74* |
| JMCC0030 intervention group | 196.79±11.34# |
Note: denotes p <0.05 compared to placebo; # denotes p <0.05 compared to model group.
TABLE 9 Effect of Streptococcus thermophilus JMCC0030 on femoral Density in rats (mg/cm)2)
| Group of | Proximal femur | Distal femur | Femur unit |
| Blank control group | 54.23±0.72 | 56.11±2.93 | 55.59±1.44 |
| Model set | 48.21±5.21* | 41.30±2.85* | 47.91±4.37* |
| JMCC0030 intervention group | 51.17±1.87# | 49.73±4.45# | 50.87±1.20# |
Note: as in table 8.
As can be seen from tables 8 and 9, the total bone density of the proximal femur, the distal femur and the femur of the model group are all significantly lower than that of the blank control group, indicating that the ovariectomized osteoporosis model is successfully established. After the prognosis of JMCC0030, the indexes are obviously improved, which shows that the JMCC0030 can improve E in blood2Concentration such that E of the run is2The concentration is close to that of a blank control group, so that the bone density of an intervention group is improved, and the osteoporosis caused by estrogen deficiency is improved, namely the streptococcus thermophilus JMCC0030 strain provided by the invention has the characteristic of improving the osteoporosis.
Although the present invention has been described in detail with reference to the above embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Shijiazhuang Junle Baoru Co Ltd
<120> Streptococcus thermophilus JMCC0030, and separation and purification method and application thereof
<130> 2020.11.26
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1398
<212> DNA
<213> Streptococcus thermophilus
<400> 1
aaggttacct caccgacttc gggtgttaca aactctcgtg gtgtgacggg cggtgtgtac 60
aaggcccggg aacgtattca ccgcggcgtg ctgatccgcg attactagcg attccgactt 120
catgtaggcg agttgcagcc tacaatccga actgagattg gctttaagag attagctcgc 180
cgtcaccgac tcgcaactcg ttgtaccaac cattgtagca cgtgtgtagc ccaggtcata 240
aggggcatga tgatttgacg tcatccccac cttcctccgg tttattaccg gcagtctcgc 300
tagagtgccc aactgaatga tggcaactaa caataggggt tgcgctcgtt gcgggactta 360
acccaacatc tcacgacacg agctgacgac aaccatgcac cacctgtcac cgatgtaccg 420
aagtaacttt ctatctctag aaatagcatc gggatgtcaa gacctggtaa ggttcttcgc 480
gttgcttcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca attcctttga 540
gtttcaacct tgcggtcgta ctccccaggc ggagtgctta atgcgttagc tgcggcactg 600
aatcccggaa aggatccaac acctagcact catcgtttac ggcgtggact accagggtat 660
ctaatcctgt tcgctcccca cgctttcgag cctcagcgtc agttacagac cagagagccg 720
ctttcgccac cggtgttcct ccatatatct acgcatttca ccgctacaca tggaattcca 780
ctctcccctt ctgcactcaa gtttgacagt ttccaaagcg aactatggtt gagccacagc 840
ctttaacttc agacttatca aaccgcctgc gctcgcttta cgcccaataa atccggacaa 900
cgctcgggac ctacgtatta ccgcggctgc tggcacgtag ttagccgtcc ctttctggta 960
agctaccgtc acagtgtgaa ctttccactc tcacacccgt tcttgactta caacagagct 1020
ttacgatccg aaaaccttct tcactcacgc ggcgttgctc ggtcagggtt gcccccattg 1080
ccgaagattc cctactgctg cctcccgtag gagtctgggc cgtgtctcag tcccagtgtg 1140
gccgatcacc ctctcaggtc ggctatgtat cgtcgcctag gtgagccatt acctcaccta 1200
ctagctaata caacgcaggt ccatcttgta gtggaacaat tgcccctttc aaataaatga 1260
catgtgtcat ccattgttat gcggtattag ctatcgtttc caatagttat cccccgctac 1320
aaggcaggtt acctacgcgt tactcacccg ttcgcaactc atccaagaag agcaagctcc 1380
tctcttcagc gttctact 1398
<210> 2
<211> 467
<212> DNA
<213> Streptococcus thermophilus
<400> 2
gaacccggat acctgactct cagcacttgt gggtgaacca taccagcacc aaggatctca 60
atccaacctg tattcttgca tacgttacat cctttaccac cacacttgaa gcatgacacg 120
tcaacctcaa cggaaggttc agtgaatggg aagtaagaag gacgcaaacg gattcgacgt 180
tctgcaccaa acattttttg aataatcatc tcaagcgttc ccttcagatc acccattgag 240
atgtttttac caacgaccaa accttcgatt tggtgaaact ggtggctgtg agtcgcatca 300
tcggtatcac gacggaaaac acgtcctggt gagatcatct taagaggacc tttagaaaaa 360
tcatgtttat caagtgtacg agcttggaca ggacttgtat gagtgcgaag caagatttct 420
tcggtaatgt agaaagtatc ttgcatatca cgaggctgga tgggtct 467
Claims (9)
1. A streptococcus thermophilus JMCC0030, characterized by: the strain is preserved in the strain preservation center of the institute of microbiology, China academy of sciences, with the preservation number of CGMCC NO.19075 and the Latin name of Streptococcus thermophilus.
2. The streptococcus thermophilus JMCC0030 as claimed in claim 1, wherein: the streptococcus thermophilus JMCC0030 is separated and screened from Xinjiang traditional fermented products.
3. Streptococcus thermophilus JMCC0030 according to claim 1 or 2, wherein: the 16SrDNA sequence of the streptococcus thermophilus JMCC0030 is as follows:
AAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGATTGGCTTTAAGAGATTAGCTCGCCGTCACCGACTCGCAACTCGTTGTACCAACCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATTACCGGCAGTCTCGCTAGAGTGCCCAACTGAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGATGTACCGAAGTAACTTTCTATCTCTAGAAATAGCATCGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCGGCACTGAATCCCGGAAAGGATCCAACACCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGAGAGCCGCTTTCGCCACCGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCCCTTCTGCACTCAAGTTTGACAGTTTCCAAAGCGAACTATGGTTGAGCCACAGCCTTTAACTTCAGACTTATCAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTCCCTTTCTGGTAAGCTACCGTCACAGTGTGAACTTTCCACTCTCACACCCGTTCTTGACTTACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCGGTCAGGGTTGCCCCCATTGCCGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTATGTATCGTCGCCTAGGTGAGCCATTACCTCACCTACTAGCTAATACAACGCAGGTCCATCTTGTAGTGGAACAATTGCCCCTTTCAAATAAATGACATGTGTCATCCATTGTTATGCGGTATTAGCTATCGTTTCCAATAGTTATCCCCCGCTACAAGGCAGGTTACCTACGCGTTACTCACCCGTTCGCAACTCATCCAAGAAGAGCAAGCTCCTCTCTTCAGCGTTCTACT。
4. the Streptococcus thermophilus JMCC0030 according to claim 3, wherein: the sequence of the Phos gene of the streptococcus thermophilus JMCC0030 is as follows:
GAACCCGGATACCTGACTCTCAGCACTTGTGGGTGAACCATACCAGCACCAAGGATCTCAATCCAACCTGTATTCTTGCATACGTTACATCCTTTACCACCACACTTGAAGCATGACACGTCAACCTCAACGGAAGGTTCAGTGAATGGGAAGTAAGAAGGACGCAAACGGATTCGACGTTCTGCACCAAACATTTTTTGAATAATCATCTCAAGCGTTCCCTTCAGATCACCCATTGAGATGTTTTTACCAACGACCAAACCTTCGATTTGGTGAAACTGGTGGCTGTGAGTCGCATCATCGGTATCACGACGGAAAACACGTCCTGGTGAGATCATCTTAAGAGGACCTTTAGAAAAATCATGTTTATCAAGTGTACGAGCTTGGACAGGACTTGTATGAGTGCGAAGCAAGATTTCTTCGGTAATGTAGAAAGTATCTTGCATATCACGAGGCTGGATGGGTCT。
5. streptococcus thermophilus JMCC0030 according to claim 1 or 2, wherein: the sequence of the Phos gene of the streptococcus thermophilus JMCC0030 is as follows:
GAACCCGGATACCTGACTCTCAGCACTTGTGGGTGAACCATACCAGCACCAAGGATCTCAATCCAACCTGTATTCTTGCATACGTTACATCCTTTACCACCACACTTGAAGCATGACACGTCAACCTCAACGGAAGGTTCAGTGAATGGGAAGTAAGAAGGACGCAAACGGATTCGACGTTCTGCACCAAACATTTTTTGAATAATCATCTCAAGCGTTCCCTTCAGATCACCCATTGAGATGTTTTTACCAACGACCAAACCTTCGATTTGGTGAAACTGGTGGCTGTGAGTCGCATCATCGGTATCACGACGGAAAACACGTCCTGGTGAGATCATCTTAAGAGGACCTTTAGAAAAATCATGTTTATCAAGTGTACGAGCTTGGACAGGACTTGTATGAGTGCGAAGCAAGATTTCTTCGGTAATGTAGAAAGTATCTTGCATATCACGAGGCTGGATGGGTCT。
6. a method for separating and purifying Streptococcus thermophilus JMCC0030 according to any one of claims 1 to 5, wherein: the separation and purification method is sequentially carried out according to the following steps:
collecting samples
Adding Xinjiang traditional fermentation products into normal saline, and fully and uniformly mixing to obtain a sample A;
② sample enrichment
Adding the sample A into an MRS liquid culture medium, and culturing at 35-40 ℃ for 62-82 h to obtain a culture solution B;
③ separating and screening strains
Taking the culture solution B, performing gradient dilution by using sterile normal saline, coating the culture solution B on an MRS solid culture medium, performing anaerobic culture for 62-82 h at the temperature of 35-40 ℃, and observing the growth condition of bacterial colonies;
after the plate has the typical bacteria, selecting a corresponding single bacterial colony C according to the bacterial colony characteristics of the standard streptococcus thermophilus;
purification of bacterial strain
And (3) inoculating the single colony C to an MRS solid culture medium, and culturing for multiple times in an aerobic environment with the culture temperature of 35-40 ℃ to obtain the streptococcus thermophilus JMCC0030 strain.
7. The separation and purification method according to claim 6, wherein: the raw materials for preparing the effective components of the MRS liquid culture medium comprise, by weight: 10 parts of casein peptone, 10 parts of beef extract, 5 parts of yeast extract, 20 parts of glucose, 5 parts of sodium acetate, 2 parts of citric acid diamine, 801 parts of tween-K2HPO42 parts of MgSO 24·7H20.2 part of O and MnSO4·7H20.05 part of O and 1000 parts of distilled water.
8. The separation and purification method according to any one of claims 6 or 7, wherein: the MRS solid culture medium is prepared by adding 15 parts of agar into 1000 parts of the MRS liquid culture medium.
9. A use of Streptococcus thermophilus JMCC0030 according to any one of claims 1 to 5, wherein: the streptococcus thermophilus JMCC0030 is compounded with the Lactobacillus bulgaricus JMCC0018 to be used for preparing fermented milk;
wherein the Lactobacillus bulgaricus JMCC0018 is preserved in the culture collection center of the institute of microbiological research of the academy of sciences of China with the preservation number of CGMCC NO. 14425.
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