CN112458013A - Streptococcus thermophilus JMCC0028, and separation and purification method and application thereof - Google Patents
Streptococcus thermophilus JMCC0028, and separation and purification method and application thereof Download PDFInfo
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- 241000194020 Streptococcus thermophilus Species 0.000 title claims abstract description 77
- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000000746 purification Methods 0.000 title claims abstract description 19
- 238000000926 separation method Methods 0.000 title claims abstract description 19
- 238000004321 preservation Methods 0.000 claims abstract description 10
- 235000021001 fermented dairy product Nutrition 0.000 claims abstract description 8
- 239000001963 growth medium Substances 0.000 claims description 21
- 239000000243 solution Substances 0.000 claims description 16
- 238000012258 culturing Methods 0.000 claims description 15
- 239000007787 solid Substances 0.000 claims description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- 230000001580 bacterial effect Effects 0.000 claims description 12
- 238000007865 diluting Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 238000003860 storage Methods 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
- 239000002504 physiological saline solution Substances 0.000 claims description 3
- YGYAWVDWMABLBF-UHFFFAOYSA-N Phosgene Chemical group ClC(Cl)=O YGYAWVDWMABLBF-UHFFFAOYSA-N 0.000 claims description 2
- 239000011248 coating agent Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims description 2
- 235000015140 cultured milk Nutrition 0.000 abstract description 35
- 238000000855 fermentation Methods 0.000 abstract description 32
- 230000004151 fermentation Effects 0.000 abstract description 32
- 239000003205 fragrance Substances 0.000 abstract description 9
- 235000014048 cultured milk product Nutrition 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 13
- 230000001953 sensory effect Effects 0.000 description 8
- 239000007788 liquid Substances 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 5
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Natural products OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
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- 102000004190 Enzymes Human genes 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
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- -1 citric acid diamine Chemical class 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
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- 229910052564 epsomite Inorganic materials 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 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
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
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- 239000006041 probiotic Substances 0.000 description 2
- 230000000529 probiotic effect Effects 0.000 description 2
- 235000018291 probiotics Nutrition 0.000 description 2
- 108010009004 proteose-peptone Proteins 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
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- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 244000199885 Lactobacillus bulgaricus Species 0.000 description 1
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- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 235000021552 granulated sugar Nutrition 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229940004208 lactobacillus bulgaricus Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
- 235000020185 raw untreated milk Nutrition 0.000 description 1
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Classifications
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- 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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Abstract
The invention discloses a streptococcus thermophilus JMCC0028, a separation and purification method and application thereof, belonging to the field of biological engineering, wherein the strain is obtained by collecting a sample from a Xinjiang traditional fermented dairy product, enriching the sample, separating the strain and purifying the strain, the strain is preserved in the strain preservation center of the institute of microbiology of China academy of sciences, and the preservation number is CGMCC NO. 19073. The streptococcus thermophilus JMCC0028 and the streptococcus thermophilus JMCC0024 are used in a matched mode, so that the fragrance (clean fragrance and no peculiar smell) and the preference degree of fermented milk are improved while the texture and the fermentation speed of the fermented milk are not influenced, and the fermented milk can be used for preparing fermented milk products.
Description
Technical Field
The invention belongs to the field of bioengineering, and relates to a strain, a screening method and application thereof, in particular to streptococcus thermophilus JMCC0028, a separation and purification method and application thereof.
Background
Streptococcus thermophilus (Streptococcus thermophilus) is one of the commonly used probiotic strains which are specified in China and can be used in food at present, and can be widely applied to the fields of food fermentation, industrial lactic acid fermentation, feed industry, medical care and the like.
The application number 201910344674.6 of the Chinese invention patent discloses Streptococcus thermophilus JMCC0024 and a separation and purification method and application thereof, the Streptococcus thermophilus (with the preservation number of CGMCC NO.16940) has the effect of improving the texture of fermented milk, can replace additives in the fermented milk and ensure the good texture of the fermented milk, but in practical application, the problem of insufficient fragrance release of fermented milk exists in fermented milk prepared by using the Streptococcus thermophilus, so that the development of a strain which is matched with the Streptococcus thermophilus JMCC0024 for use, does not influence the texture of the fermented milk and can improve the problem of insufficient fragrance release of the fermented milk is urgently needed.
Disclosure of Invention
The invention aims to provide a streptococcus thermophilus JMCC0028 which is used in combination with streptococcus thermophilus JMCC0024 to improve the fragrance (clean and odorless) and preference of fermented milk without influencing the texture and the fermentation speed of the fermented milk;
another objective of the invention is to provide a method for separating and purifying Streptococcus thermophilus JMCC 0028;
it is still another object of the present invention to provide the use of the streptococcus thermophilus JMCC0028 as described above.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the streptococcus thermophilus JMCC0028 is deposited in the culture collection center of the institute of microbiology of China academy of sciences, the collection address is No. 3 of Xilu No.1 of Beijing republic of Chaoyang, the collection date is 2019, 12 and 3 days, and the collection number is CGMCC NO. 19073.
As a limitation of the invention, the 16Sr RNA sequence of the Streptococcus thermophilus JMCC0028 is as follows:
CGGCTGGCTCCAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGATTGGCTTTAAGAGATTAGCTCGCCGTCACCGACTCGCAACTCGTTGTACCAACCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATTACCGGCAGTCTCGCTAGAGTGCCCAACTGAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGATGTACCGAAGTAACTTTCTATCTCTAGAAATAGCATCGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTTCGGCACTGAATCCCGGAAAGGATCCAACACCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGAGAGCCGCTTTCGCCACCGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCCCTTCTGCACTCAAGTTTGACAGTTTCCAAAGCGAACTATGGTTGAGCCACAGCCTTTAACTTCAGACTTATCAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTCCCTTTCTGGTAAGCTACCGTCACAGTGTGAACTTTCCACTCTCACACCCGTTCTTGACTTACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCGGTCAGGGTTGCCCCCATTGCCGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTATGTATCGTCGCCTAGGTGAGCCATTACCTCACCTACTAGCTAATACAACGCAGGTCCATCTTGTAGTGGAGCAATTGCCCCTTTCAAATAAATGACATGTGTCATCCATTGTTATGCGGTATTAGCTATCGTTTCCAATAGTTATCCCCCGCTACAAGGCAGGTTACCTACGCGTTACTCACCCGTTCGCAACTCATCCAAGAAGAGCAAGCTCCTCTCTTCAGCGTTCTACT。
as another limitation of the invention, the Phos gene sequence of Streptococcus thermophilus JMCC0028 is as follows:
GCGGAACCCCGATCACCCTGACATCTCAGCACTTGTGGGTGACCATACCAGCACCAAGGATCTCAATCCAACCTGTCTTCTTGCATACGTTACATCCTTTACCACCACACTTGAAGCATGACACGTCAACCTCAACGGAAGGTTCAGTGAATGGGAAGTAAGAAGGACGCAAACGGATTTGACGTTCTGCACCAAACATTTTTTGAATAATCATCTCAAGCGTTCCCTTCAGATCACCCATTGAGATGTTTTTACCAACGACCAAACCTTCGATTTGGTGAAACTGGTGGCTGTGAGTCGCATCATCGGTATCACGACGGAAAACACGTCCTGGTGAGATCATCTTAAGAGGACCTTTAGAAAAATCATGTTTATCAAGTGTACGAGCTTGGACAGGACTTGTATGAGTGCGAAGCAAGATTTCTTCGGTAATGTAGAAAGTATCTTGCATATCACGAGCAGGATGA。
the invention also provides a separation and purification method of the streptococcus thermophilus JMCC0028, which is carried out according to the following steps:
(ii) sample Collection
Adding Xinjiang traditional fermented dairy products into physiological saline, and mixing to obtain a collected sample;
② sample enrichment
Adding the collected sample into an MRS liquid culture medium for culture to obtain a culture solution;
③ separation of bacterial strains
Taking a culture solution, diluting the culture solution by using sterile normal saline, coating the diluted culture solution on an MRS solid culture medium, and culturing the MRS solid culture medium in an anaerobic environment;
after the plate has the typical bacteria, selecting a single colony P according to the colony characteristics of the standard streptococcus thermophilus;
purification of bacterial strain
And (3) selecting a single colony P, streaking and inoculating the single colony P to another MRS solid culture medium, culturing in an aerobic environment, then selecting a single colony Q on the sterile plate, and continuously streaking and inoculating the single colony Q to another MRS solid culture medium, and continuously culturing for multiple times until the colony forms in the culture medium are consistent, thus obtaining the streptococcus thermophilus JMCC0028 strain.
As a limitation of the invention, after the strain is purified, the JMCC0028 strain and sterile 50% glycerol are put into a strain storage tube, and are uniformly mixed and stored at-70 ℃.
The invention also provides an application of the streptococcus thermophilus JMCC0028, and the streptococcus thermophilus JMCC0028 is used for preparing a fermented dairy product.
As a limitation of the invention, Streptococcus thermophilus JMCC0028 is used in combination with Streptococcus thermophilus JMCC0024 for preparing fermented dairy products.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the technical progress that:
the streptococcus thermophilus JMCC0028 and the streptococcus thermophilus JMCC0024 are used in a matched mode, so that the fragrance (clean fragrance and no peculiar smell) and the preference degree of fermented milk can be improved while the texture and the fermentation speed of the fermented milk are not influenced;
secondly, the method for separating and purifying the streptococcus thermophilus JMCC0028 is simple;
the streptococcus thermophilus JMCC0028 provided by the invention has good fermentation characteristics in the application process, has high fermentation speed, and is beneficial to saving the production time and the production cost; the pH value corresponding to the fermentation end point is low (about 4.1), and the fermentation liquid has good tolerance to a strong acid environment, is beneficial to the survival of the fermentation liquid in an extreme environment of a human digestive tract, and can better play a probiotic role; the fermented milk prepared by fermenting the fermented milk has good viscosity; the fermented milk prepared by the method and the streptococcus thermophilus JMCC0024 through matched fermentation has the advantages of good texture, clean aroma, no peculiar smell, proper acidity and viscosity in mouth and good overall preference.
Detailed Description
Example 1 method for separation and purification of Streptococcus thermophilus JMCC0028
The separation and purification method of streptococcus thermophilus JMCC0028 is carried out according to the following steps:
(ii) sample Collection
Adding 25mL of Xinjiang traditional fermented dairy product into 250mL of physiological saline, and mixing to obtain a collected sample;
② sample enrichment
Taking 2mL of collected sample, adding the sample into 80mL of MRS liquid culture medium, and culturing at 37 ℃ for 70h to obtain a culture solution B;
③ separation of bacterial strains
Taking 1mL of culture solution, diluting with sterile normal saline by 10 times, and respectively making 10 times of the culture solution with sterile normal saline-1、10-2、10-3、10-4、10-5Diluting by times in a gradient manner to obtain bacterial suspensions with different concentrations;
taking an MRS solid culture medium, melting, pouring the MRS solid culture medium into a sterilized culture dish to prepare a sterile flat plate, and sucking 0.1mL of bacterial suspensions with different concentrations to respectively coat the sterile flat plate after the MRS solid culture medium is cooled and completely solidified;
culturing at 35 deg.C under anaerobic condition for 65 hr;
after the plate has the typical bacteria, selecting a corresponding single bacterial colony P 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 P, streaking and inoculating the selected single colony P to another sterile plate, culturing for 65 hours in an aerobic environment at 35 ℃, then selecting a single colony Q on the sterile plate, continuously streaking and inoculating to another sterile plate, continuously culturing for three times until the colony forms in a culture medium are consistent, observing the cell forms of the strains through a microscope to obtain streptococcus thermophilus, selecting the streptococcus thermophilus by an inoculating loop, inoculating to a liquid MRS culture medium, and culturing for 24 hours at 37 ℃ to obtain a final pure culture strain;
fifthly, preservation of the strain
Respectively taking 800 mu L of pure culture strain and sterile 50% glycerol, placing the pure culture strain and the sterile 50% 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 embodiment, the MRS liquid medium is composed of the following raw materials: 8g of casein peptone, 12g of beef extract, 6g of yeast extract, 16g of glucose, 6g of sodium acetate, 2.5g of citric acid diamine, 801.2 g of tween-K2HPO4 2.5g、MgSO4·7H2O 0.1g、MnSO4·7H20.06g of O and 1000mL of distilled water.
The MRS solid culture medium consists of the following raw materials: 12g of casein peptone, 12g of beef extract, 6g of yeast extract, 16g of glucose, 6g of sodium acetate, 2.5g of citric acid diamine, 801.2 g of tween-K2HPO4 2.5g、MgSO4·7H2O 0.1g、MnSO4·7H20.06g of O, 17g of agar and 1000mL of distilled water.
Examples 2 to 6 Streptococcus thermophilus and method for separating and purifying the same
Examples 2 to 6 are a streptococcus thermophilus strain and a separation and purification method thereof, wherein the specific steps of the separation and purification method are similar to those of example 1, and the differences are only that: the relevant parameters are different, and are specifically shown in table 1.
Table 1 specific steps and parameters of separation and purification methods of examples 2 to 6
Example 7 fermentation characteristics of Streptococcus thermophilus
Respectively inoculating the JMCC0028 and W2-W6 strains, the JMCC0024 strain (with the preservation number of CGMCC NO.16940), the Lactobacillus bulgaricus JMCC0018 (with the preservation number of CGMCC NO.14425) and the Streptococcus thermophilus JMCC16 (with the preservation number of CGMCC NO.11672) screened in the embodiments 1-6 into a liquid MRS culture medium to culture at 37 ℃ for 48h to obtain corresponding first generation strain culture solution;
respectively adding 1mL of corresponding first generation 1mL of strain culture solution into 9mL of skim milk, and culturing for 16h to obtain corresponding second generation strain culture solution;
adding 1mL of corresponding second generation strain culture solution into 9mL of skimmed milk, culturing for 16h to obtain corresponding third generation strain seed solution, and freezing at-80 deg.C.
Respectively preparing fermented milk by taking corresponding third-generation strain seed liquid, and specifically operating as follows: weighing 1kg of raw milk and 70g of white granulated sugar as base materials, inoculating 30g of corresponding third generation strain seed liquid (the inoculation mode is shown in table 2), fermenting at 42 ℃ for 6h to prepare fermented milk, and respectively testing the fermentation speed, the viscosity test and the sensory test of each set of fermented milk.
Table 2 preparation of a summary of fermented milk parameters
First, fermentation speed (pH value after fermentation for 6 h), viscosity test
Counting the pH value of the groups A1-A10 after fermentation for 6h (because the pH value does not reach 4.5 after fermentation for 6h, the production time is prolonged and the production cost is increased because of actual production needs); after fermenting for 6h, demulsifying by using a stirrer (800rpm, 2.5min), standing at 4 ℃ overnight, measuring the viscosity of the fermented milk after standing overnight at room temperature for 4h by using a viscometer (rotor: LV-03; rotating speed: 60 rpm; end condition: 30s), and repeating three times to obtain an average value, wherein the test results of each test item are shown in the following table:
TABLE 3 fermentation speed (pH after 6h fermentation) and viscosity test results
| Group of | pH value after fermentation for 6h | Viscosity measurement (cP) after 6h fermentation |
| A1 | 4.41 | 1539 |
| A2 | 4.40 | 1520 |
| A3 | 4.32 | 1580 |
| A4 | 4.44 | 948 |
| A5 | 5.10 | 880 |
| A6 | 5.01 | 960 |
| A7 | 5.20 | 1480 |
| A8 | 4.80 | 1390 |
| A9 | 4.49 | 1250 |
| A10 | 4.48 | 1580 |
As can be seen from the above table, different strains have complex synergistic and competitive relationships in the process of being used with the Streptococcus thermophilus JMCC0024, namely in the process of fermenting fermented milk, so that the characteristics of the fermented milk are affected differently. Compared with the fermented milk of group A10 (JMCC 0024), some strains increased the fermentation speed of the fermented milk but affected the viscosity of the fermented milk, such as group A4; some strains both inhibit the fermentation rate and influence the viscosity of fermented milks, such as groups A5, A6, A7, A8; some strains can improve the fermentation speed of the fermented milk and have little influence on the viscosity of the fermented milk, such as A1, A2 and A3 groups (the pH is below 4.5 after fermentation for 6 hours, and the viscosity is above 1500 cP).
Second, sensory test
15 professional product research and dairy tasters tasted the fermented milks prepared in groups A1-A10, evaluated the flavor and condition of the fermented milks according to the sensory evaluation criteria in Table 4, and counted the average of the sensory evaluations in groups A1-A10, the results are shown in Table 5:
TABLE 4 sensory test evaluation criteria
TABLE 5 summary of sensory test results
| Group of | Overall preference | Fragrance | Viscosity of the oil | Peculiar smell |
| A1 | 7.24 | 5.94 | 7.29 | 1.72 |
| A2 | 4.32 | 2.77 | 5.59 | 4.57 |
| A3 | 6.05 | 3.12 | 7.22 | 2.12 |
| A4 | 5.32 | 2.40 | 6.51 | 3.89 |
| A5 | 5.01 | 2.03 | 6.32 | 3.01 |
| A6 | 4.89 | 2.95 | 5.54 | 3.87 |
| A7 | 5.01 | 2.49 | 3.78 | 4.23 |
| A8 | 6.02 | 2.33 | 5.79 | 4.49 |
| A9 | 6.08 | 5.12 | 6.28 | 2.49 |
| A10 | 6.21 | 3.15 | 7.32 | 2.01 |
As can be seen from the above table, different strains combined with JMCC0024 during the fermentation process of the fermented milk produce different sensory taste characteristics, wherein the overall preference of the a1, A3, A8, a9 and a10 groups is better (more than 6 points), the a1 group has the best effect (more than 7 points), the viscosity of the fermented milk is better maintained, the fragrance of the fermented milk is obvious, and the off-flavor is less.
In summary, the fermentation characteristic test results of the streptococcus thermophilus provided by the invention are good (including fermentation speed, acidity, viscosity test and sensory evaluation), and the above experimental results show that the streptococcus thermophilus obtained by the separation and purification method of the invention can be used for preparing fermented milk products, and the effect of the streptococcus thermophilus obtained by the separation and purification method of the invention is better when the streptococcus thermophilus is used in combination with JMCC0024, wherein the streptococcus thermophilus JMCC0028 strain obtained by the separation and purification in example 1 is the best, and the performance of the streptococcus thermophilus JMCC0028 obtained by the separation and purification in example 1 is tested and protected.
Example 8 bacteriological characterization of Streptococcus thermophilus JMCC0028
Basic characteristics
Example 1 the screened streptococcus thermophilus JMCC0028 basic features are shown in table 6:
TABLE 6 basic characteristics of Streptococcus thermophilus JMCC0028
| Experimental project | Gram stain | Oxidase enzyme | Cell morphology | Contact enzyme | Contact enzyme |
| Results of the experiment | Positive for | - | Spherical shape | - | - |
Second, sugar fermentation characteristic experiment
Selecting a single colony of streptococcus thermophilus JMCC0028, carrying out plate streaking, culturing for 24h at 37 ℃, carrying out passage once, taking bacterial suspension, respectively inoculating the bacterial suspension into different sugar fermentation tubes (the specific content in the sugar fermentation tubes is shown in Table 7), culturing for 48h at 37 ℃, and observing color change, wherein the specific result is shown in Table 7:
TABLE 7 identification of Streptococcus thermophilus JMCC0028
Note: "+" indicates fermentation utilization; "-" indicates no fermentative utilization.
Thirdly, molecular biology identification
The streptococcus thermophilus JMCC0028 strain screened in example 1 is identified as streptococcus thermophilus by DNA extraction, PCR amplification and 16SrRNA sequencing, and NCBI website blast is finally determined as streptococcus thermophilus.
The 16Sr RNA sequence of streptococcus thermophilus JMCC0028 is as follows:
CGGCTGGCTCCAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGATTGGCTTTAAGAGATTAGCTCGCCGTCACCGACTCGCAACTCGTTGTACCAACCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATTACCGGCAGTCTCGCTAGAGTGCCCAACTGAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGATGTACCGAAGTAACTTTCTATCTCTAGAAATAGCATCGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTTCGGCACTGAATCCCGGAAAGGATCCAACACCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGAGAGCCGCTTTCGCCACCGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCCCTTCTGCACTCAAGTTTGACAGTTTCCAAAGCGAACTATGGTTGAGCCACAGCCTTTAACTTCAGACTTATCAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTCCCTTTCTGGTAAGCTACCGTCACAGTGTGAACTTTCCACTCTCACACCCGTTCTTGACTTACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCGGTCAGGGTTGCCCCCATTGCCGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTATGTATCGTCGCCTAGGTGAGCCATTACCTCACCTACTAGCTAATACAACGCAGGTCCATCTTGTAGTGGAGCAATTGCCCCTTTCAAATAAATGACATGTGTCATCCATTGTTATGCGGTATTAGCTATCGTTTCCAATAGTTATCCCCCGCTACAAGGCAGGTTACCTACGCGTTACTCACCCGTTCGCAACTCATCCAAGAAGAGCAAGCTCCTCTCTTCAGCGTTCTACT。
the gene sequence of the Phos of Streptococcus thermophilus JMCC0028 is as follows:
GCGGAACCCCGATCACCCTGACATCTCAGCACTTGTGGGTGACCATACCAGCACCAAGGATCTCAATCCAACCTGTCTTCTTGCATACGTTACATCCTTTACCACCACACTTGAAGCATGACACGTCAACCTCAACGGAAGGTTCAGTGAATGGGAAGTAAGAAGGACGCAAACGGATTTGACGTTCTGCACCAAACATTTTTTGAATAATCATCTCAAGCGTTCCCTTCAGATCACCCATTGAGATGTTTTTACCAACGACCAAACCTTCGATTTGGTGAAACTGGTGGCTGTGAGTCGCATCATCGGTATCACGACGGAAAACACGTCCTGGTGAGATCATCTTAAGAGGACCTTTAGAAAAATCATGTTTATCAAGTGTACGAGCTTGGACAGGACTTGTATGAGTGCGAAGCAAGATTTCTTCGGTAATGTAGAAAGTATCTTGCATATCACGAGCAGGATGA。
the streptococcus thermophilus JMCC0028 is preserved in a strain preservation center of the institute of microbiology of China academy of sciences, the preservation address is No. 3 of Xilu No.1 of Beijing Korean-Yangzhou, the preservation date is 12 months and 3 days in 2019, and the preservation number is CGMCC NO. 19073.
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 JMCC0028, and separation and purification method and application thereof
<130> 2020-11-25
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1410
<212> DNA
<213> Streptococcus thermophilus (Streptococcus thermophilus)
<400> 1
cggctggctc caaaggttac ctcaccgact tcgggtgtta caaactctcg tggtgtgacg 60
ggcggtgtgt acaaggcccg ggaacgtatt caccgcggcg tgctgatccg cgattactag 120
cgattccgac ttcatgtagg cgagttgcag cctacaatcc gaactgagat tggctttaag 180
agattagctc gccgtcaccg actcgcaact cgttgtacca accattgtag cacgtgtgta 240
gcccaggtca taaggggcat gatgatttga cgtcatcccc accttcctcc ggtttattac 300
cggcagtctc gctagagtgc ccaactgaat gatggcaact aacaataggg gttgcgctcg 360
ttgcgggact taacccaaca tctcacgaca cgagctgacg acaaccatgc accacctgtc 420
accgatgtac cgaagtaact ttctatctct agaaatagca tcgggatgtc aagacctggt 480
aaggttcttc gcgttgcttc gaattaaacc acatgctcca ccgcttgtgc gggcccccgt 540
caattccttt gagtttcaac cttgcggtcg tactccccag gcggagtgct taatgcgtta 600
gcttcggcac tgaatcccgg aaaggatcca acacctagca ctcatcgttt acggcgtgga 660
ctaccagggt atctaatcct gttcgctccc cacgctttcg agcctcagcg tcagttacag 720
accagagagc cgctttcgcc accggtgttc ctccatatat ctacgcattt caccgctaca 780
catggaattc cactctcccc ttctgcactc aagtttgaca gtttccaaag cgaactatgg 840
ttgagccaca gcctttaact tcagacttat caaaccgcct gcgctcgctt tacgcccaat 900
aaatccggac aacgctcggg acctacgtat taccgcggct gctggcacgt agttagccgt 960
ccctttctgg taagctaccg tcacagtgtg aactttccac tctcacaccc gttcttgact 1020
tacaacagag ctttacgatc cgaaaacctt cttcactcac gcggcgttgc tcggtcaggg 1080
ttgcccccat tgccgaagat tccctactgc tgcctcccgt aggagtctgg gccgtgtctc 1140
agtcccagtg tggccgatca ccctctcagg tcggctatgt atcgtcgcct aggtgagcca 1200
ttacctcacc tactagctaa tacaacgcag gtccatcttg tagtggagca attgcccctt 1260
tcaaataaat gacatgtgtc atccattgtt atgcggtatt agctatcgtt tccaatagtt 1320
atcccccgct acaaggcagg ttacctacgc gttactcacc cgttcgcaac tcatccaaga 1380
agagcaagct cctctcttca gcgttctact 1410
<210> 2
<211> 467
<212> DNA
<213> Streptococcus thermophilus (Streptococcus thermophilus)
<400> 2
gcggaacccc gatcaccctg acatctcagc acttgtgggt gaccatacca gcaccaagga 60
tctcaatcca acctgtcttc ttgcatacgt tacatccttt accaccacac ttgaagcatg 120
acacgtcaac ctcaacggaa ggttcagtga atgggaagta agaaggacgc aaacggattt 180
gacgttctgc accaaacatt ttttgaataa tcatctcaag cgttcccttc agatcaccca 240
ttgagatgtt tttaccaacg accaaacctt cgatttggtg aaactggtgg ctgtgagtcg 300
catcatcggt atcacgacgg aaaacacgtc ctggtgagat catcttaaga ggacctttag 360
aaaaatcatg tttatcaagt gtacgagctt ggacaggact tgtatgagtg cgaagcaaga 420
tttcttcggt aatgtagaaa gtatcttgca tatcacgagc aggatga 467
Claims (7)
1. The streptococcus thermophilus JMCC0028 is preserved in the culture collection center of the institute of microbiology of academy of sciences of China with the preservation number of CGMCC NO. 19073.
2. The Streptococcus thermophilus JMCC0028 of claim 1, wherein the 16Sr RNA sequence of the Streptococcus thermophilus JMCC0028 is as follows:
CGGCTGGCTCCAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGATTGGCTTTAAGAGATTAGCTCGCCGTCACCGACTCGCAACTCGTTGTACCAACCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATTACCGGCAGTCTCGCTAGAGTGCCCAACTGAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGATGTACCGAAGTAACTTTCTATCTCTAGAAATAGCATCGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTTCGGCACTGAATCCCGGAAAGGATCCAACACCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGAGAGCCGCTTTCGCCACCGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCCCTTCTGCACTCAAGTTTGACAGTTTCCAAAGCGAACTATGGTTGAGCCACAGCCTTTAACTTCAGACTTATCAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTCCCTTTCTGGTAAGCTACCGTCACAGTGTGAACTTTCCACTCTCACACCCGTTCTTGACTTACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCGGTCAGGGTTGCCCCCATTGCCGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTATGTATCGTCGCCTAGGTGAGCCATTACCTCACCTACTAGCTAATACAACGCAGGTCCATCTTGTAGTGGAGCAATTGCCCCTTTCAAATAAATGACATGTGTCATCCATTGTTATGCGGTATTAGCTATCGTTTCCAATAGTTATCCCCCGCTACAAGGCAGGTTACCTACGCGTTACTCACCCGTTCGCAACTCATCCAAGAAGAGCAAGCTCCTCTCTTCAGCGTTCTACT。
3. the Streptococcus thermophilus JMCC0028 according to claim 1 or 2, wherein the Phos gene sequence of the Streptococcus thermophilus JMCC0028 is:
GCGGAACCCCGATCACCCTGACATCTCAGCACTTGTGGGTGACCATACCAGCACCAAGGATCTCAATCCAACCTGTCTTCTTGCATACGTTACATCCTTTACCACCACACTTGAAGCATGACACGTCAACCTCAACGGAAGGTTCAGTGAATGGGAAGTAAGAAGGACGCAAACGGATTTGACGTTCTGCACCAAACATTTTTTGAATAATCATCTCAAGCGTTCCCTTCAGATCACCCATTGAGATGTTTTTACCAACGACCAAACCTTCGATTTGGTGAAACTGGTGGCTGTGAGTCGCATCATCGGTATCACGACGGAAAACACGTCCTGGTGAGATCATCTTAAGAGGACCTTTAGAAAAATCATGTTTATCAAGTGTACGAGCTTGGACAGGACTTGTATGAGTGCGAAGCAAGATTTCTTCGGTAATGTAGAAAGTATCTTGCATATCACGAGCAGGATGA。
4. the method for separating and purifying Streptococcus thermophilus JMCC0028 according to any one of claims 1 to 3, wherein the method comprises the following steps:
(ii) sample Collection
Adding Xinjiang traditional fermented dairy products into physiological saline, and mixing to obtain a collected sample;
② sample enrichment
Adding the collected sample into an MRS liquid culture medium for culture to obtain a culture solution;
③ separation of bacterial strains
Taking a culture solution, diluting the culture solution by using sterile normal saline, coating the diluted culture solution on an MRS solid culture medium, and culturing the MRS solid culture medium in an anaerobic environment;
after the plate has the typical bacteria, selecting a single colony P according to the colony characteristics of the standard streptococcus thermophilus;
purification of bacterial strain
And (3) selecting the single colony P, streaking and inoculating the single colony P to another MRS solid culture medium, culturing in an aerobic environment, then selecting the single colony Q on the sterile plate, continuously streaking and inoculating the single colony Q to another MRS solid culture medium, and continuously culturing for at least three times until the colony forms in the culture medium are consistent, thus obtaining the streptococcus thermophilus JMCC0028 strain.
5. The method for separating and purifying Streptococcus thermophilus JMCC0028 as claimed in claim 4, wherein after the strain is purified, the JMCC0028 strain and sterile glycerol are placed in a strain storage tube, and the strain is stored at-70 ℃ after being mixed uniformly.
6. A use of Streptococcus thermophilus JMCC0028 as claimed in any of claims 1 to 3 wherein: the streptococcus thermophilus JMCC0028 is used for preparing fermented dairy products.
7. The use of Streptococcus thermophilus JMCC0028 according to claim 6, wherein: the streptococcus thermophilus JMCC0028 and the streptococcus thermophilus JMCC0024 are matched for preparing the fermented dairy product.
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| CN116286521A (en) * | 2023-03-14 | 2023-06-23 | 微康益生菌(苏州)股份有限公司 | Streptococcus thermophilus with endogenous sweetening effect, strain combination and application thereof |
| CN116286521B (en) * | 2023-03-14 | 2023-10-24 | 微康益生菌(苏州)股份有限公司 | Streptococcus thermophilus with endogenous sweetening effect, strain combination and application thereof |
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