CN117402771A - Pediococcus pentosaceus GT-303 and application thereof in preparation of low-salt soybean paste - Google Patents
Pediococcus pentosaceus GT-303 and application thereof in preparation of low-salt soybean paste Download PDFInfo
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- CN117402771A CN117402771A CN202310947936.4A CN202310947936A CN117402771A CN 117402771 A CN117402771 A CN 117402771A CN 202310947936 A CN202310947936 A CN 202310947936A CN 117402771 A CN117402771 A CN 117402771A
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- pediococcus pentosaceus
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- 241000191996 Pediococcus pentosaceus Species 0.000 title claims abstract description 46
- 235000010469 Glycine max Nutrition 0.000 title claims abstract description 40
- 244000068988 Glycine max Species 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims description 5
- 238000000855 fermentation Methods 0.000 claims abstract description 13
- 230000004151 fermentation Effects 0.000 claims abstract description 13
- 241000894006 Bacteria Species 0.000 claims abstract description 9
- 238000004321 preservation Methods 0.000 claims abstract description 4
- 150000003839 salts Chemical class 0.000 claims description 8
- 241000192001 Pediococcus Species 0.000 claims description 7
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 4
- 238000002425 crystallisation Methods 0.000 claims description 2
- 230000008025 crystallization Effects 0.000 claims description 2
- 241000519995 Stachys sylvatica Species 0.000 abstract description 9
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 21
- 239000007788 liquid Substances 0.000 description 16
- 230000001580 bacterial effect Effects 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 10
- 238000012258 culturing Methods 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 230000000844 anti-bacterial effect Effects 0.000 description 7
- 230000004913 activation Effects 0.000 description 6
- 238000000034 method Methods 0.000 description 6
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- 229920001817 Agar Polymers 0.000 description 4
- 241000191940 Staphylococcus Species 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 238000009630 liquid culture Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 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 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000035882 stress Effects 0.000 description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 239000004278 EU approved seasoning Substances 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 235000021107 fermented food Nutrition 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 235000011194 food seasoning agent Nutrition 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 239000006916 nutrient agar Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
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- 235000013555 soy sauce Nutrition 0.000 description 2
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- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241001147736 Staphylococcus capitis Species 0.000 description 1
- 241000192087 Staphylococcus hominis Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 238000013124 brewing process Methods 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- KLOIYEQEVSIOOO-UHFFFAOYSA-N carbocromen Chemical compound CC1=C(CCN(CC)CC)C(=O)OC2=CC(OCC(=O)OCC)=CC=C21 KLOIYEQEVSIOOO-UHFFFAOYSA-N 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000010411 cooking Methods 0.000 description 1
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- 230000007547 defect Effects 0.000 description 1
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- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 1
- 230000015784 hyperosmotic salinity response Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 235000014659 low sodium diet Nutrition 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000000399 optical microscopy Methods 0.000 description 1
- 230000008650 pH stress Effects 0.000 description 1
- 229940023462 paste product Drugs 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 235000015424 sodium Nutrition 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
- A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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Abstract
The invention discloses Pediococcus pentosaceus GT-303 and application thereof in preparing low-salt soybean paste, wherein Pediococcus pentosaceus (Pediococcus pentosaceus) GT-303 is preserved in China Center for Type Culture Collection (CCTCC) No. M20231052 in the 6 th month and 19 th year of 2023, the preservation address is China university of Wuhan and Wuhan, and the Pediococcus pentosaceus GT-303 is applied to fermentation production of low-salt soybean paste, the generation of white spots in the low-salt soybean paste is reduced, spoilage bacteria in the low-salt soybean paste are inhibited, and the Pediococcus pentosaceus has good effects in the aspects of improving the safety and the attractiveness of the low-salt soybean paste and wide application prospect.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to Pediococcus pentosaceus GT-303 and application thereof in preparation of low-salt soybean paste.
Background
The soybean paste is prepared from soybean and flour as main raw materials, adding microorganisms such as Aspergillus oryzae to make Daqu, and adding saline water with a certain concentration for temperature control fermentation to obtain semi-fluid food. As a traditional fermented food, the soybean paste has unique color, smell, taste and body state, is salty, sweet and palatable, is rich in nutrition, is often used for cooking various dishes or dipping foods, and is deeply favored by people.
Excessive intake of salt (sodium) is an important risk factor for diseases such as hypertension and diabetes. Most of soybean pastes sold in the market have a salt content of more than 10%, and the high-salt soybean paste not only limits the use amount of the product, but also conflicts with the low-salt diet which is popular at present. With the improvement of living conditions, high-salt diets are increasingly not advocated, the salt content of traditional fermented seasonings is reduced, and the production of low-salt or even salt-free fermented seasonings is widely focused by people.
The low-salt soybean paste product is a necessary trend of upgrading and updating the product, and the low-salt inoculation process is used for making the product more green and healthy, enabling the flavor to be plump, fermenting the soybean paste in a low-salt environment, enabling the microorganism to grow more vigorous, and being beneficial to improving the flavor of the soybean paste.
However, the soybean paste often has white crystals with irregular shapes during production and storage, commonly known as white spots. And with the extension of the shelf life of soybean paste commodity, white spots also have a tendency to increase, and the appearance of soybean paste finished products is seriously influenced. In the fermentation process of soybean paste, materials are decomposed into polypeptide and amino acid under the action of protease, peptidase and other enzymes, tyrosine in the amino acid is only 0.045% in solubility, so that the tyrosine is easy to separate out in the brewing process, white spots are generated, the appearance quality of the soybean paste is seriously influenced, the consumption confidence of common people is reduced, and huge economic loss is brought to enterprises.
At present, pediococcus pentosaceus is mostly used in the fields of fermented foods, biological preservatives and the like, and Pediococcus pentosaceus is also approved as an edible fungus. However, the method has not been found to be applied to the low-salt soybean paste for solving the problem of white spots and inhibiting spoilage bacteria in the fermentation process of the low-salt soybean paste.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description summary and in the title of the application, to avoid obscuring the purpose of this section, the description summary and the title of the invention, which should not be used to limit the scope of the invention.
The present invention has been made in view of the above and/or problems occurring in the prior art.
Therefore, the invention aims to overcome the defects in the prior art and provide Pediococcus (Pediococcus) GT-303 which is preserved in China Center for Type Culture Collection (CCTCC) No. M20231052 and has a preservation address of university of Chinese, wuhan and Wuhan, wherein Pediococcus (Pediococcus) GT-303 grows in an environment with 10 percent NaClw/w and pH of 2.5, and has strong stress resistance.
It is a further object of the present invention to overcome the deficiencies of the prior art and to provide the use of Pediococcus pentosaceus (Pediococcus pentosaceus) GT-303 in the preparation of a low salt soybean paste.
As a preferred embodiment of the application according to the invention, wherein: the Pediococcus pentosaceus (Pediococcus pentosaceus) GT-303 has the ability to significantly reduce the tyrosine crystallization white point in low salt soybean paste.
As a preferred embodiment of the application according to the invention, wherein: the Pediococcus pentosaceus (Pediococcus pentosaceus) GT-303 has the capability of inhibiting spoilage bacteria in the fermentation process of low-salt soybean paste.
The invention has the beneficial effects that:
the Pediococcus pentosaceus GT-303 is applied to low-salt soybean paste fermentation production, reduces the generation of white spots in the low-salt soybean paste, inhibits spoilage bacteria in the low-salt soybean paste, has good effects in improving the safety and the attractive appearance of the low-salt soybean paste, and has wide application prospects.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 shows colony morphology of Pediococcus pentosaceus on MRS nutrient agar, MRS-CaCO in the examples of the invention 3 Nutrient agar and optical microscopy.
FIG. 2 is a phylogenetic tree of identification of Pediococcus pentosaceus strains in the practice of the invention.
FIG. 3 is a graph showing growth data of Pediococcus pentosaceus under sodium chloride stress in examples of the present invention.
FIG. 4 is a graph showing the pH stress growth data of Pediococcus pentosaceus in examples of the present invention.
FIG. 5 is a graph showing white spots of low-salt soybean paste in the example of the present invention, wherein (a) is experimental group 1 (fermented for 7 days), (b) is experimental group 2 (fermented for 21 days), (c) is control group 1 (fermented for 7 days), and (d) is control group 2 (fermented for 21 days).
FIG. 6 is a diagram of the antibacterial activity of Pediococcus pentosaceus in an example of the invention, wherein (a) is a diagram of the antibacterial activity of human staphylococcus, and (b) is a diagram of the antibacterial activity of human staphylococcus.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The invention provides Pediococcus pentosaceus (Pediococcus) GT-303 which is preserved in China Center for Type Culture Collection (CCTCC) No. M20231052 in the year 2023 and the month 19, and the preservation address is China university of Wuhan.
Example 1
Separation of Pediococcus pentosaceus:
the sample is prepared from soy sauce mash of northeast farmhouse by adopting a five-point method, and is placed in a sterile EP pipe after being fully and uniformly mixed and stored in a refrigerator at-80 ℃.
Weighing soy sauce sample 5.0g under aseptic condition, mixing with 45mL sterile physiological saline, shaking in shaking table for 2 hr to disperse microbial cells, and making into 10 -1 Dilutions were made and serial dilutions were performed.
Respectively taking 0.1mL of dilution of 10 -5 、10 -6 And 10 -7 Is inoculated into MRS-CaCO 3 Solid culture medium (peptone 10g, yeast powder 5g, beef extract 10g, diammonium hydrogen citrate 2g, glucose 20g, tween-80 1mL, sodium acetate 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.58g, manganese sulfate 0.25g, agar 18g, distilled water 1L, after sterilizing, cooling MRS culture medium to 45-50 ℃, adding sterilized calcium carbonate 20g, fully mixing, pouring into a flat plate to obtain) coating, placing the flat plate in an anaerobic incubator at 37 ℃ and a common incubator to culture for 48h respectively, observing and picking colonies with transparent rings around, and repeatedly streaking and purifying to obtain pure strain.
The glycerol pipe is preserved, and the bacterial liquid and 50% of glycerol are mixed according to the following ratio of 1:1 are mixed evenly and stored in a refrigerator at the temperature of minus 80 ℃.
The stored strains were activated three times and subjected to physiological and biochemical experiments.
Colony morphology, colony size, color, shape, colony edge, protrusion and transparency were observed and recorded as in FIG. 1.
It can be seen that transparent rings are generated around the picked colonies, and the color of the colonies is mainly white and milky; the appearance is mainly round, the surface is smooth and moist, the texture is uniform, and the picking is easy. Gram staining is purple and spherical, and negative strains are obtained in catalase reaction, nitrate reduction experiment, gelatin liquefaction experiment, hydrogen sulfide experiment, indole experiment and motility experiment.
Example 2
Molecular biological identification of Pediococcus pentosaceus:
culturing the activated strain to logarithmic phase, collecting bacterial liquid 1mL, centrifuging 8000r/min to collect bacterial cells, adding sterile water 100 μl, mixing to give cell concentration of about 10 5 mu.L.
10 mu L of the mixture is added into 90 mu L of sterile water to dilute 10 times, and 10 is prepared 5 、10 4 、10 3 、10 2 And 10 bacteria solutions with concentration of mu.L, 3 mu.L of each dilution was inoculated in MRS-CaCO 3 On the solid plate, colony growth was observed.
And selecting a strain with a calcium-dissolving ring for identification.
Extracting genome of the strain to be tested by using a DNA extraction kit, carrying out PCR amplification by using bacterial 16S rRNA sequence universal primers (27F and 1492R), sequencing the PCR amplification product by using a gold-only biotechnology company, carrying out BLAST homology search on the spliced sequence result in a GenBank database, and constructing a phylogenetic tree by using MEGA. Phylogenetic tree was constructed using the adjacency method (neighbor-joining) in MEGA, as shown in fig. 2.
The nucleotide sequence (shown as seq_1) of Pediococcus pentosaceus GT_303 in the invention is:
AACCCTGTCCACCTTAGACGGCTAGCTCCTAAAAGGTTACCCCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGTTTTAAGAGATTAGCTTAACCTCGCGGTTTCGCGACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACT TAATGCTGGCAACTAGTAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACCTCTAATCTCTTAGACTGTCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCTTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGATTACTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGTAATCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCCAGTTTCCAATGCACTTCTTCGGTTGAGCCGAAAGGCTTTCACATTAGACTTAAAAGACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGGTTAAATACCGTCACTGGGTAAACAGTTACTC。
example 3
Determination of stress resistance of Pediococcus pentosaceus:
directly pouring the frozen strain in glycerol pipe into 100mLMRS liquid culture medium (1% soybean peptone, 2% glucose, 0.5% yeast powder), culturing at 37deg.C with shaking table 120rpm for 48 hr, inoculating the cultured bacterial liquid into 100mL fresh MRS liquid at 3%, culturing at 37deg.C with shaking table 120rpm to OD 600 =0.7, considered to complete activation.
The activated strain was inoculated to MRS liquid medium with NaCI content of 5%, 6%, 7%, 8%, 9%, 10% NaCl (w/w) at an inoculum size of 3%, and cultured at 37℃for 24 hours to obtain fermentation broth. The absorbance value (OD) was measured at a wavelength of 600nm 600nm ) The control was tested 3 times in parallel with a blank MRS broth with corresponding NaCI content.
Inoculating the activated strain at 3% inoculum size into MRS liquid culture medium with pH value of 2.5, 3.5, 4.5, 5.5, 6.5, respectively, and culturing at 37deg.C for 24 hr to obtain fermentation broth, and measuring absorbance (OD) at wavelength of 600nm 600nm ) The control was a blank MRS broth with a pH value corresponding to each of them, and the samples were tested in parallel for 3 times.
As a result of the measurement, as shown in FIG. 3, pediococcus pentosaceus GT-303 was found to grow under 10% NaCl conditions, showing good salt tolerance.
As a result of the measurement, as shown in FIG. 4, pediococcus pentosaceus GT-303 was found to grow at pH 2.5, showing good acid resistance.
Example 4
Pediococcus pentosaceus has the ability to reduce white spots in soybean paste:
directly pouring the frozen strain in the glycerol pipe into 1Culturing in 00mL MRS liquid culture medium at 37deg.C shaking table 120rpm for 48 hr, inoculating the cultured bacterial liquid into 100mL fresh MRS liquid at 3%, and culturing at 37deg.C shaking table 120rpm to OD 600 =0.7, considered to complete activation.
Centrifuging the activated strain at 8000r/min and 4deg.C for 10min, removing upper medium, washing with sterile physiological saline (0.85% by mass) twice, and adjusting to 10 6 cfu/ml of the bacterial suspension is added into soybean paste, and fermentation is carried out for 7 days and 21 days at the temperature of 30 ℃.
Control groups 1, 2 were not supplemented with Pediococcus pentosaceus GT-303; experiment groups 1 and 2A fermentation experiment of low-salt soybean paste was performed with Pediococcus pentosaceus GT-303 added.
The measurement results are shown in FIG. 5, in which white spots are significantly reduced after sterilization of the low salt soybean paste added with Pediococcus pentosaceus GT_303.
Example 5
Application of Pediococcus pentosaceus in inhibiting putrefying bacteria:
the frozen strain (Pediococcus pentosaceus GT-303) in the glycerol pipe is directly poured into 100mL MRS liquid culture medium (1% soybean peptone, 2% glucose, 0.5% yeast powder), cultured for 48h at 37 ℃ by shaking table 120rpm, then the cultured bacterial liquid is inoculated into 100mL fresh MRS liquid according to the proportion of 3%, and cultured to OD at 37 ℃ by shaking table 120rpm 600 =0.7, considered to complete activation.
The activated Pediococcus pentosaceus GT-303 is inoculated in MRS culture solution with an inoculum size of 3 percent, and incubated for 16 to 18 hours at 37 ℃.
The culture broth was then centrifuged at 8000 Xg for 30min at 4 ℃. The supernatant was collected and subjected to a 0.22 μm filter to obtain a cell-free fermentation supernatant. The antibacterial activity is detected by a double-layer agar plate method.
Activation of indicator bacteria: directly pouring the frozen strain (staphylococcus hominis) in glycerol pipe into 100mL LB liquid medium, culturing at 30deg.C with shaking table 120rpm for 48h, inoculating the cultured bacterial liquid into 100mL fresh LB liquid at 3%, culturing at 30deg.C with shaking table 120rpm to OD 600 =0.7, considered to complete activation.
Directly pouring frozen strain (Staphylococcus cephalomasum) in glycerol tubeCulturing in 100mL LB liquid medium at 30deg.C shaking table 120rpm for 48 hr, inoculating the cultured bacterial liquid into 100mL fresh LB liquid at 3%, and culturing at 30deg.C shaking table 120rpm to OD 600 =0.7, considered to complete activation.
The activated indicator bacteria staphylococcus humanus and staphylococcus capitis are respectively inoculated in a liquid LB culture medium for culturing at the constant temperature of 30 ℃ for 12 hours.
The bacteriostasis method comprises the following steps: the antibacterial property of the lactobacillus fermentation supernatant was measured by the international double-layer agar plate diffusion method, and the pediococcus pentosaceus gt_303 was studied. The bottom layer culture medium is about 10mL and 1.8% of agar culture medium, after drying for 30min, a proper amount of sterile oxford cup is uniformly placed, then 20mL of LB agar culture medium at 50 ℃ is poured, 0.1% of indicator fungus suspension upper layer is inoculated, after drying for 1h, the oxford cup is pulled out, 100 mu L of cell-free supernatant is added in the air, the mixture is diffused for 2h in a refrigerator at 4 ℃, then the mixture is placed in a constant temperature incubator at 30 ℃ for culture, and the diameters of inhibition zones are respectively measured.
The measurement results are shown in Table 1 and FIG. 6, and Pediococcus pentosaceus GT-303 has antibacterial effect on both bacteria.
TABLE 1 determination of antibacterial Properties of Pediococcus pentosaceus
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, and it should be covered in the scope of the present invention.
Claims (5)
1. Pediococcus pentosaceus (Pediococcus) GT-303 is preserved in China Center for Type Culture Collection (CCTCC) No. M20231052 at the 6 th month and 19 th year of 2023, and the preservation address is China university of Wuhan.
2. Pediococcus pentosaceus (Pediococcus) GT-303 of claim 1, which grows in 10% NaClw/w pH 2.5 environment, has strong stress resistance.
3. Use of pediococcus pentosaceus (GT-303) according to claim 1 or 2 for the preparation of a low salt soybean paste.
4. The use according to claim 3, characterized in that: the Pediococcus pentosaceus (Pediococcus pentosaceus) GT-303 has the ability to significantly reduce the tyrosine crystallization white point in low salt soybean paste.
5. The use according to claim 3, characterized in that: the Pediococcus pentosaceus (Pediococcus pentosaceus) GT-303 has the capability of inhibiting spoilage bacteria in the fermentation process of low-salt soybean paste.
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