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CN118460416B - A Staphylococcus aureus strain with odor reduction function and its application - Google Patents

A Staphylococcus aureus strain with odor reduction function and its application Download PDF

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CN118460416B
CN118460416B CN202410616500.1A CN202410616500A CN118460416B CN 118460416 B CN118460416 B CN 118460416B CN 202410616500 A CN202410616500 A CN 202410616500A CN 118460416 B CN118460416 B CN 118460416B
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strain
staphylococcus
methylphenol
carbon source
pyrazine
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CN118460416A (en
Inventor
伊奎鑫
李世文
姚进明
刘仁杰
章金梅
唐云
李文慧
裴中强
覃德叁
左宗辉
邱丽丽
李艳英
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Zhejiang Gengshengtang Ecological Agriculture Co ltd
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Zhejiang Gengshengtang Ecological Agriculture Co ltd
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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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L5/00Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20Removal of unwanted matter, e.g. deodorisation or detoxification
    • A23L5/28Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00Food compositions, function of food ingredients or processes for food or foodstuffs
    • 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
    • C12R2001/44Staphylococcus

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
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  • Organic Chemistry (AREA)
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  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Nutrition Science (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Food Science & Technology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明公开了一株具有异味消减功能的葡萄球菌菌株及其应用,属于生物工程技术领域。通过微生物筛选及鉴定等方法,在麻竹笋传统发酵过程中获得一株生长状态良好、国家公认的可用于食品添加的、并对发酵产生的异味主效成分4‑甲基苯酚(p‑Cresol)以及吡嗪有较好降解能力的葡萄球菌Staphylococcussp.MB‑Z4。

The invention discloses a Staphylococcus strain with odor reduction function and its application, belonging to the field of bioengineering technology. Through microbial screening and identification methods, a Staphylococcus sp. MB-Z4 with good growth status, nationally recognized as a food additive, and good degradation ability for the main odor components 4-methylphenol (p-Cresol) and pyrazine produced by fermentation is obtained in the traditional fermentation process of bamboo shoots.

Description

Staphylococcus strain with odor reducing function and application thereof
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a staphylococcus strain with an odor reducing function and application thereof.
Background
The fresh bastose is short in shelf life and relatively single in taste, most of bastose is processed in a pickling mode for facilitating eating and carrying of consumers, different pickling processes also endow the bastose pickled products with different flavors and tastes, and the variety of pickled foods is greatly enriched. Along with the continuous establishment and expansion of the bamboo shoot processing enterprises, the processing modes of the bamboo shoot products are diversified and gradually increased, but manufacturers gradually find that unpleasant peculiar smell substances are generated in the fermentation process of the fresh bamboo shoot products, and the unpleasant peculiar smell substances are often accompanied with bad sensory characteristics such as astringent taste and the like, so that most consumers are difficult to adapt and accept, and the unpleasant peculiar smell substances are not present in the processing process of other bamboo products. In addition, the generation of fermentation peculiar smell does not have exact reasons and control means, the uncertain factors of the generation of the peculiar smell are large, and the fermentation peculiar smell of the bamboo shoots cannot be controlled by a plurality of manufacturers, so that the fermented bamboo shoot products cannot normally flow into the market, a large amount of raw materials are wasted, and the operation cost is increased severely.
Therefore, the problem of the fermentation peculiar smell of the bast shoots becomes a bottleneck problem in the processing process, so that the further development of the bast shoot processing industry is greatly limited. Therefore, if the peculiar smell substances generated in the processing process of the bast bamboo shoots can be lightened and finally eliminated, the application range of the bast bamboo shoots in the market can be enlarged, the digestion speed is accelerated, and the problems of backlog and waste of the bast bamboo shoots of peasant households are solved.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the application and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description of the application and in the title of the application, which may not be used to limit the scope of the application.
The present invention has been made in view of the above and/or problems occurring in the prior art.
Therefore, the invention aims to screen an endogenous microorganism strain from fermented bamboo shoots, overcomes the defects in the prior art and provides a staphylococcus strain with an odor reducing function, and is characterized in that the staphylococcus strain is staphylococcus aureus.MB-Z4 and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2024451 and the preservation date of 2024 for 03 months and 18 days.
A preferred embodiment of the staphylococcus strain of the invention is one in which the staphylococcus strain is capable of growing with 4-methylphenol as the sole carbon source.
A preferred embodiment of the staphylococcus strain of the invention is one in which the staphylococcus strain is capable of growing with pyrazine as the sole carbon source.
It is a further object of the present invention to overcome the deficiencies of the prior art and to provide a use of a staphylococcus strain.
As a preferred embodiment of the use according to the invention, the staphylococcus strain may be used in the field of food fermentation or for reducing off-flavors in processed foods.
The invention has the beneficial effects that:
the staphylococcus Staphylococcus aureus MB-Z4 which has good growth state, can be used for food addition and has good degradation capability on the main active components of peculiar smell generated by fermentation, namely 4-methylphenol (p-Cresol) and pyrazine is obtained in the traditional fermentation process of the bamboos through microorganism screening and identification and other methods.
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 the strain of the present invention on LB medium.
FIG. 2 shows the results of gram staining of the strains of the invention alone.
FIG. 3 shows the result of gram staining of a strain of the invention mixed with E.coli.
FIG. 4 shows a phylogenetic tree of MB-Z4 strains according to the present invention.
FIG. 5 shows the detection of the ability of the strain of the invention to degrade off-flavor substances.
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 embodiment of the invention provides a staphylococcus strain with an odor reducing function, which is staphylococcus aureus.MB-Z4 and is preserved in China center for type culture collection (CCTCC for short, address: eight channel 299 No. in Wuchang area of Wuhan, hubei province, and post code 430072) in the 18 th year of 2024, and the preservation number is CCTCC NO: M2024451.
The raw materials used in the examples of the present invention are commercially available as usual unless otherwise specified.
Example 1
This example is the isolation of a strain, comprising the steps of:
taking 1.0g of a traditional 7-day-fermented and pickled phyllostachys praecox shoot sample, placing the sample in a mortar, adding 10mL of sterile water, fully grinding, filtering insoluble substances by using sterile gauze, centrifuging at a low temperature and a low speed for 3-5 min, and taking 1mL of supernatant in a brand-new sterile EP tube to obtain a fermented bamboo shoot treatment liquid;
Then, carrying out gradient dilution on the fermentation liquor by 10-10 7 times, selecting three dilution gradient fermentation liquor of 10 5、106 and 10 7, respectively taking 100 mu L of dilution liquor, and coating the dilution liquor on a common LB solid plate;
culturing the coated flat plate in a 37 ℃ constant temperature incubator for 24-36 hours in an inverted mode until single colony with proper shape and size is grown;
and (3) selecting a plurality of single colonies with normal growth morphology, and performing shake culture in an LB liquid medium for 24 hours to obtain bacterial liquid.
A common inorganic salt basic culture medium is prepared, 1.5mL of inorganic salt culture medium is divided into a plurality of sterilized 2mL of EP tubes, and 4-methylphenol and pyrazine with the content of 1% are respectively added as unique carbon sources, wherein the 4-methylphenol is not dissolved in the inorganic salt culture medium, so that the 4-methylphenol can be seen to be paved above the inorganic salt liquid culture medium in an oil drop shape.
Different strains to be detected which are preserved in LB liquid culture medium are centrifuged at 12000rpm for 2min at low temperature, the supernatant is removed to retain thalli, the thalli are washed for 2 times by sterilized normal saline, and then 1mL of sterilized normal saline is used for resuspension of the thalli, so that the influence of the culture medium on the measurement result is avoided.
Each strain to be tested was inoculated separately into 2 unique carbon source selection media containing 4-methylphenol and pyrazine while only sterile water was inoculated as a negative control group. Each EP tube was tightly sealed with an airtight plastic film to avoid air leakage. After the EP tubes are placed in a 37 ℃ constant temperature shaking table for culturing for 48-72 hours, OD 600 values are measured to detect the growth condition of the thalli in each tube, and the existence condition of upper oil drops in the 4-methylphenol selective medium is observed. Strains were selected that were able to grow in both 4-methylphenol and pyrazine.
The observation of oil drops shows that the oil drops in the negative control group are not disappeared, part of the oil drops still exist in the culture tubes corresponding to different strains, and the OD 600 value is smaller than 0.03, so that the strain can not grow by using 4-methylphenol as the sole carbon source or has extremely low utilization rate. Also for the strain grown in pyrazine, if the OD 600 value was less than 0.03, it was considered that the strain could not grow or had very low availability with pyrazine as the sole carbon source.
Finally, strain MB-Z4 which can efficiently utilize 4-methylphenol and can maintain an excellent growth state in pyrazine is screened out.
Example 2
This example is the identification of strains, comprising the steps of:
1. Colony morphology
The morphological characteristics of the strain MB-Z4 are shown in figures 1-3 by referring to the Bojie's handbook of bacteriology of the system and the handbook of identification of the common bacterial system, and the strain is shown as an opaque circular bulge with a smooth surface, a golden yellow color, a white color or a lemon color with a diameter of 2-3mm, and has regular edges, positive gram staining, spherical thallus, single thallus, paired thallus or irregular heap-shaped thallus on an LB solid culture medium.
2. 16S rDNA sequence amplification and analysis
And extracting genome DNA by using a bacterial genome DNA extraction kit as a template for PCR reaction. The bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') were used to amplify the strain ' S16S rDNA sequence.
The reaction system was 2x PhantaMaster Mix 25. Mu.L, 1. Mu.L of DNA template, 2. Mu.L of forward primer, 2. Mu.L of reverse primer and distilled water to a volume of 20. Mu.L. The PCR reaction was performed at 95℃for 3min, denaturation at 95℃for 15s, annealing at 55℃for 15s, extension at 72℃for 50s,30 cycles, and final at 72℃for 10min.
After the PCR products were cut and purified, they were sequenced.
Sequencing results were as follows:
5’
-GGCGTGTCTATACATGCAAGTCGAGCGAACGGATAAGGAGCTTGCTCCT TTGAAGTTAGCGGGGGACGGGTGAGTAACACGTGGGTAACCTACCTATAAGACTGGGATAACTTCGGGAAACCGGAGCTAATACCGGATAACATTTAGAACCGCATGGGTCTAAATTGAAAGATGGCTTTGCTATCACTTATTGATGGACCCGCGCCGTATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGACGATACGTAGCCGACCTGAGAAGGGGATCGGCCACCCTGGAACTGAAACACGGTCCCCACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGGGAAAGCCTGACGGAGCAACGCCGCGGGAGGGATGAAGGGTTTCGGCTCGTAAAACTCTGGTATTAGGGAAAAACAAATGGGTAAGTAACTGTGCACATCTTGACGGGACCTAATCACAAAAACACGCCTAACTACGTGCCACCAGCCGCGGTAATACGTAGGTGGGAAGCGTTATCCGCAATTATTGTGGGTAAAGCGCGCGTAGGCGGGTTCTTATGTCTGATGTGAAAGACCACGGCTCAACCGCGTAGGGTCATTGTGAACTGTGAAACTTGAGAGCGCAAGAGAGAAGAGTGATTCCATGTGTAGCGGGGAGATGTGCAGAGATATGTAGGAACACCAGAGTGGAAAGCGACTCTCTGTGCTGTGACTGAGACTGAGGTGCGAGAGAGCGTGGATCGAACAGGAGTATGATACCCCTGGTGAGTCCACGCCGCCATACGATGAGTGCT-3'
The 16S rDNA sequencing results of the strains were aligned in the NCBI website with BLASTN sequences, which indicated that the test strain had a sequence similarity of 97.18% with the 16S rDNA sequence of StaphylococcusxylosusMg 16-1 (LN 554884.1) (SEQ ID NO: OR 946361).
A suitable strain 16S rDNA sequence was selected, and a phylogenetic tree was constructed using MEGA 7.0 software and the Neighbor-Joining (NJ) method, and the results are shown in FIG. 4.
3. Physiological and biochemical experiments and unique carbon source utilization experiments
In order to finally identify the species of the strain to be tested, a series of physiological and biochemical experiment identifications are carried out on the strain by referring to Bonji system bacteriology handbook, wherein the physiological and biochemical experiment identifications comprise a thixotropic enzyme experiment, a VP experiment, a urease hydrolysis experiment, a beta-galactosidase hydrolysis experiment, a beta-glucosidase hydrolysis experiment, an optimal pH determination experiment, an optimal growth temperature determination and an LB NaCl tolerance experiment, and the experimental results are shown in table 1.
The only carbon source utilization experiments were selected from glucose, D-xylose, D-fructose, mannose, sucrose, mannitol, melibiose, trehalose, arginine and raffinose, and the results of the only carbon source utilization experiments are shown in Table 2.
In tables 1 and 2, "+" represents a positive test result, and "-" represents a negative test result.
TABLE 1 physiological and biochemical test results of strains
TABLE 2 results of experiments on unique carbon sources of strains
And combining a physiological and biochemical experiment and a molecular identification result, and determining that the peculiar smell degradation strain MB-Z4 screened by the experiment is Staphylococcus aureus.
Example 3
The example is a degradation experiment of the strain on the peculiar smell substances.
Since 4-methylphenol is present in the aqueous medium as water-insoluble oil droplets on the liquid surface, the degradation capacities of 4-methylphenol and pyrazine of the strain are determined by using the strain growth state measurement and the presence of 4-methylphenol, and specifically comprising the following steps:
The MB-Z4 strain washed twice with physiological saline and resuspended is respectively inoculated into BSM inorganic salt liquid culture medium containing 1% 4-methylphenol and 1% pyrazine according to the inoculation amount of 2%, and after strict sealing, the strain is subjected to shaking culture for 72 hours at 180rpm in a 37 ℃ constant temperature shaking table, meanwhile, a group with only the same amount of physiological saline is taken as a negative control group, and after the strain is cultured for 72 hours, the existence state of oil drops is observed.
The experimental procedure and results are shown in fig. 5.
Wherein, (a) is the condition of inoculating MB-Z4 into a culture medium of a sole carbon source of 4-methylphenol for 0h culture;
(b) The culture medium is a blank 4-methylphenol only carbon source culture medium, and the culture condition is 0 h;
(c) Inoculating MB-Z4 into a pyrazine sole carbon source culture medium, and culturing for 0 h;
(d) The culture medium is a blank pyrazine sole carbon source culture medium, and the culture condition is 0 h;
(e) Inoculating MB-Z4 into a culture medium of a unique carbon source of 4-methylphenol, and culturing for 72 hours;
(f) The culture medium is a blank 4-methylphenol only carbon source culture medium, and the culture condition is 72 hours;
(g) Inoculating MB-Z4 into a pyrazine sole carbon source culture medium, and culturing for 72 hours;
(h) The medium is the only carbon source medium for blank pyrazine and is cultivated for 72 hours.
It can be seen that after 72 hours of culture, the oil drops of the negative control group still exist, but the oil drops of the inoculated MB-Z4 group disappear, the state of the culture medium is turbid, the OD 600 value is 0.36, which indicates that the strain can grow by taking 4-methylphenol as the sole carbon source, the culture medium inoculated with pyrazine is not turbid, the culture medium inoculated with the strain has thallus for growth, and the OD 600 value is 0.61, which indicates that the strain can grow by taking pyrazine as the sole carbon source.
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 (3)

1. The Staphylococcus strain (Staphylococcus sp.) with the peculiar smell reducing function is characterized in that the Staphylococcus strain is MB-Z4 and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2024451 and the preservation date of 2024 and 03 months and 18 days.
2. The staphylococcus strain of claim 1, wherein the staphylococcus strain is capable of growing with 4-methylphenol as the sole carbon source.
3. The staphylococcal strain according to claim 1 wherein the staphylococcal strain is capable of growth with pyrazine as the sole carbon source.
CN202410616500.1A 2024-05-17 2024-05-17 A Staphylococcus aureus strain with odor reduction function and its application Active CN118460416B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106190924A (en) * 2016-08-17 2016-12-07 江南大学 One plant height produces the clostridium tyrobutyricum of 4 methylphenols
CN107129954A (en) * 2016-06-27 2017-09-05 内蒙古大学 A kind of staphylococcus of degradation of phenol

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101898260B1 (en) * 2017-11-20 2018-09-14 대한민국 Bacillus amyloliquefacience having antibacterial activity and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107129954A (en) * 2016-06-27 2017-09-05 内蒙古大学 A kind of staphylococcus of degradation of phenol
CN106190924A (en) * 2016-08-17 2016-12-07 江南大学 One plant height produces the clostridium tyrobutyricum of 4 methylphenols

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