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 PDF

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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
soybean paste
low
salt
pediococcus
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赵国忠
王萌
姚云平
乔赫梁
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Tianjin University of Science and Technology
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/20Bacteria; Culture media therefor
    • C12N1/205Bacterial isolates
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, 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
    • A23L11/00Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/50Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/01Bacteria 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

Pediococcus pentosaceus GT-303 and application thereof in preparation of low-salt soybean paste
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.
CN202310947936.4A 2023-07-31 2023-07-31 Pediococcus pentosaceus GT-303 and application thereof in preparation of low-salt soybean paste Pending CN117402771A (en)

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