CN115093989B - Streptococcus salivarius QS-20 with antioxidant activity and application thereof - Google Patents
Streptococcus salivarius QS-20 with antioxidant activity and application thereof Download PDFInfo
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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Abstract
The invention discloses streptococcus salivarius QS-20 with antioxidant activity and application thereof. The class name of the streptococcus salivarius QS-20 isStreptococcus salivariusThe strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC 7.456, and can effectively remove DPPH free radicals, hydrogen peroxide and other oxidizing substances; the bacterium has good acid-producing acid-resistant property and cholate-resistant capability, is beneficial to the field planting of the bacterium in intestinal tracts, and has good inhibitory activity on various pathogenic bacteria such as escherichia coli, bacillus subtilis, staphylococcus aureus, salmonella and pseudomonas aeruginosa. Therefore, the streptococcus salivarius QS-20 has wide application fields.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to streptococcus salivarius QS-20 with antioxidant activity and application thereof.
Background
Human milk is rich in microorganisms (including bacteria, viruses and fungi) which play an important role not only in the start of the neonatal intestinal immune system, the development of the infant intestinal immune function and the programmed course, but also in protecting infants from diseases and infections during growth. Currently, microorganisms that have been isolated and identified from breast milk are mainly staphylococcus, streptococcus, enterococcus, lactobacillus, bifidobacterium, leuconostoc, and the like. Among them, staphylococci, streptococci, bifidobacteria and lactic acid bacteria are dominant flora in breast milk, and have important probiotic effects. Therefore, the research and development of streptococcus in human milk is of great significance.
Disclosure of Invention
The invention aims to provide streptococcus salivarius QS-20 with antioxidant activity and application thereof, and the streptococcus salivarius QS-20 has antioxidant activity, acid production characteristics, bile salt resistance and antibacterial capacity.
In order to achieve the aim of the invention, the invention is realized by adopting the following technical scheme:
the invention provides a streptococcus salivarius QS-20 with antioxidant activity, wherein the streptococcus salivarius QS-20 is classified asStreptococcus salivariusThe method is preserved in China general microbiological culture Collection center, address: the preservation number is CGMCC 7.456, and the preservation date is that the North Chen Xili No.1 yard No. 3 in the Chaoyang district of Beijing city: 2022, 1, 13; the viability test result of the biological material is: survival.
Furthermore, the streptococcus salivarius QS-20 is a white raised colony with smooth surface, the diameter of the colony is 0.2-1mm, the cells are spherical or oval, the diameter is not more than 1 mu m, and the cells are arranged in a chain shape.
Further, the streptococcus salivarius QS-20 has antioxidant activity, acid production characteristics, cholate resistance and antibacterial capacity.
Further, the nucleotide sequence of the streptococcus salivarius QS-20 is shown as SEQ ID No.1
The invention also provides an application of the streptococcus salivarius QS-20 in preparing an antioxidant.
Further, the acting time of the antioxidant is 12-36 hours.
Furthermore, the streptococcus salivarius QS-20 achieves an antioxidation effect by removing DPPH free radicals and hydrogen peroxide.
The invention also provides an application of the streptococcus salivarius QS-20 in preparing a starter.
Further, the fermentation time of the starter is 0-3h.
The invention also provides application of the streptococcus salivarius QS-20 in preparing a bacteriostatic agent.
Further, the bacteriostat can inhibit escherichia coli, bacillus subtilis, staphylococcus aureus, salmonella and pseudomonas aeruginosa.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention separates probiotics from breast milk, takes DPPH clearance rate as a screening index to obtain streptococcus salivarius QS-20 with better antioxidation capability, and the probiotics have good antioxidation activity and can effectively remove DPPH free radicals, hydrogen peroxide and other oxidation substances; also has good acid-producing and acid-resisting properties, and provides basis for the application of the modified starch in fermented foods. In addition, streptococcus salivarius QS-20 has bile salt resistance, is beneficial to colonization of intestinal tracts, and has good inhibitory activity on various pathogenic bacteria such as escherichia coli, bacillus subtilis, staphylococcus aureus, salmonella and pseudomonas aeruginosa. Therefore, the streptococcus salivarius QS-20 has wide application fields.
Drawings
FIG. 1 is an electrophoretogram of the 16S rDNA amplification PCR product of a part of the strain.
FIG. 2 is a phylogenetic tree analysis of QS-20 strains.
FIG. 3 is a colony morphology and cell morphology diagram of Streptococcus salivarius QS-20.
FIG. 4 shows the growth curve and the acidogenesis curve of Streptococcus salivarius QS-20.
FIG. 5 shows DPPH radical scavenging effect of Streptococcus salivarius QS-20.
FIG. 6 shows the acid resistance curve of Streptococcus salivarius QS-20.
FIG. 7 shows a graph of the bile salt resistance of Streptococcus salivarius QS-20.
FIG. 8 shows the inhibitory effect of Streptococcus salivarius QS-20 on bacteria.
Detailed Description
The technical scheme of the invention is further described in detail by combining the following specific examples.
In the following examples, unless otherwise specified, all experimental methods used are conventional and all materials, reagents, etc. are commercially available from biological or chemical reagent companies.
Example 1: separation screening and identification of streptococcus salivarius QS-20
1. Separation and identification
Fresh breast milk was diluted with physiological saline at various concentrations and spread on MRS solid medium, and cultured upside down at 37℃for 48 hours. Selecting smooth white or milky single colony of thallus, gram staining, microscopic examination, selecting rod-shaped gram positive bacteria, and streaking and purifying on MRS solid culture medium for 2-3 times. And (3) selecting single bacterial colonies subjected to streak purification, inoculating the single bacterial colonies to an MRS liquid culture medium, continuously culturing for 24-48 hours at 37 ℃, mixing bacterial liquid and sterilized glycerol according to the ratio of 1:1, and placing the mixed bacterial liquid and sterilized glycerol in a refrigerator at-80 ℃ for later use.
(1) Extracting genome DNA by using a root genome kit;
(2) The 16S rDNA PCR amplification adopts a general primer,
upstream primer 27F: GAGAGTTTGATCCTGGCTCAG the number of the individual pieces of the plastic,
downstream primer 1492R: TACGGCTACCTTGTTACGAC.
The PCR reaction system was 50ul: taq Reaction Buffer (10×) 5ul,PCR Dye 5ul,dNPTs 1ul,Taq Dymerase 0.5ul,DNA template 2ul,27F 2ul,1492R2ul, water was added to make up to 50ul.
The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5min; cycling 30 times (95 ℃ C., 30s denaturation; 55 ℃ C., 30s annealing; 72 ℃ C., 90s extension); extending at 72 deg.C for 7min, and extending at 20 deg.C for 3min.
(3) Sequencing of PCR amplified products
The PCR products were detected by 1% agarose gel electrophoresis and sent to Qingdao department of Biotechnology for sequencing. The sequences were aligned for homology to the GenBank database by the BLAST program.
16 bacteria were isolated from three different breast milk samples by the transparent circle method, and 16S rDNA amplification was performed to obtain a target band of about 1500 bp (FIG. 1), the nucleotide sequence of which is shown in SEQ ID No. 1.
Sequencing the target band, and performing phylogenetic tree analysis, wherein the QS-20 strain and the QS-20 strain can be known from the phylogenetic tree of FIG. 2Streptococcus salivariusThe relatedness is recent and therefore it belongs to streptococcus salivarius.
Performing strain preservation on the strain QS-20, wherein the preservation unit of the streptococcus salivarius QS-20 is as follows: china general microbiological culture Collection center (CGMCC); address: the institute of microbiology, national academy of sciences, north chen xi lu 1, 3, the region of the morning sun in beijing; preservation date: 2022, 1, 13; the classification is named: streptococcus salivariusStreptococcus salivarius，The preservation number is CGMCC 7.456, and the viability detection result of the biological material is as follows: survival.
2. Morphological observation
Colony morphology and gram-stained cell morphology were observed for the QS-20 strain and the results are shown in FIG. 3. The white raised colony of QS-20 strain has smooth surface, colony diameter of 0.2-1mm, spherical or oval cell diameter not exceeding 1 micron, chain arrangement and deposition growth in liquid culture medium. The QS-20 strain can generate acid to form a transparent ring on the MRS culture medium added with calcium carbonate, the result of gram staining shows that the QS-20 strain is gram-positive bacteria, meanwhile, the catalase experiment of the strain is negative, the result further shows that the QS-20 strain is streptococcus in the gram-positive bacteria, and the result is consistent with the analysis result of the evolutionary tree.
Example 2: biochemical Properties of Streptococcus salivarius QS-20
1. Growth and acid production curves of Streptococcus salivarius QS-20 strain
Referring to FIG. 4, the QS-20 strain can rapidly enter the logarithmic phase in a short time, has strong adaptability and short adaptation period, enters the exponential phase 5-15h, and enters the stationary phase after 5-15h, and has no obvious decay phase within 52 h. In the exponential growth phase, the streptococcus salivarius QS-20 has stronger acid production and obviously reduced pH, so that the streptococcus salivarius QS-20 is suitable to be used as a starter.
2. Determination of antioxidant Activity of Streptococcus salivarius QS-20 Strain
After the DPPH free radical is reduced by the antioxidant, the DPPH free radical becomes stable DPPH molecule, the color is changed from dark purple to light yellow, and the purple intensity of the DPPH is inversely proportional to the antioxidant activity of the sample. DPPH has a stable structure, and if the DPPH can be eliminated, the DPPH shows that the tested strain has the chain reaction effects of reducing hydrogen peroxide and eliminating various free radicals. Streptococcus salivarius QS-20 strain was inoculated into MRS broth liquid medium, and the antioxidant activity of fermentation supernatant was measured at different culture times.
As shown in FIG. 5, it can be seen that the QS-20 strain has a very high DPPH clearance rate, the clearance rate reaches more than 70% after 12 hours, and the clearance rate reaches more than 80% after 36 hours, thus showing a strong antioxidant activity.
3. Acid resistance determination of Streptococcus salivarius QS-20 Strain
The QS-20 strain is inoculated into MRS broth liquid culture medium with pH of 3.0 for 3 hours and 5 hours, the liquid culture medium without thalli and with pH of 3.0 is used as a blank, OD600nm is measured, the culture time is used as an abscissa, the absorbance value is used as a slave coordinate, and a broken line trend chart is drawn.
As can be seen from FIG. 6, the absorbance gradually increased within 3 hours, indicating that the QS-20 strain survived and grew; the absorbance at 5h did not change much compared to 3h, indicating that the growth of the strain was inhibited. From this, it was found that the strain had a maximum survival tolerance time of 0 to 3 hours under acidic conditions.
4. Determination of bile salt resistance of Streptococcus salivarius QS-20 Strain
Inoculating QS-20 strain into MRS broth liquid culture medium containing different bile salt concentrations (0.1%, 0.2%, 0.3%) for culturing for 3 and 5 hours, taking liquid culture medium containing no thallus and different concentration of bile salt as blank, measuring absorbance at OD600nm, taking culturing time as abscissa, absorbance value as slave coordinate, and drawing a broken line trend chart.
As shown in FIG. 7, streptococcus salivarius QS-20 survived and grew within 5 hours, and after 3 hours, the high concentration of bile salts had a certain inhibitory effect on the growth of the strain.
Example 3: antibacterial ability analysis of Streptococcus salivarius QS-20
Culturing Escherichia coli, bacillus subtilis, staphylococcus aureus, salmonella and Pseudomonas aeruginosa, and detecting antibacterial activity of Streptococcus salivarius QS-20 on the above strains.
The results in FIG. 8 show that Streptococcus salivarius QS-20 strain has inhibitory activity against Escherichia coli, bacillus subtilis, staphylococcus aureus, salmonella and Pseudomonas aeruginosa, wherein the inhibitory activity against Bacillus subtilis and Pseudomonas aeruginosa is stronger.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
<110> Qingdao university of agriculture
<120> Streptococcus salivarius QS-20 having antioxidant activity and application thereof
<141> 2022-04-08
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gagtgtgaga gtggaaagtt cacactgtga cggtagctta ccagaaaggg acggctaact 480
acgtgccagc agccgcggta atacgtaggt cccgagcgtt gtccggattt attgggcgta 540
aagcgagcgc aggcggtttg ataagtctga agttaaaggc tgtggctcaa ccatagttcg 600
ctttggaaac tgtcaaactt gagtgcagaa ggggagagtg gaattccatg tgtagcggtg 660
aaatgcgtag atatatggag gaacaccggt ggcgaaagcg gctctctggt ctgtaactga 720
cgctgaggct cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt 780
aaacgatgag tgctaggtgt tggatccttt tcgggattca gtgccgcagc taacgcatta 840
agcactccgc ctggggagta cgaccgcaag gttgaaactc aaaggaattg acgggggccc 900
gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt 960
gacatcccga tgctatttct agagatagaa agttacttcg gtacatcggt gacaggtggt 1020
gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080
ccctattgtt agttgccatc attcagttgg gcactctagc gagactgccg gtaataaacc 1140
ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg 1200
ctacaatggt tggtacaacg agttgcgagt cggtgacggc aagctaatct cttaaagcca 1260
atctcagttc ggattgtagg ctgcaactcg cctacatgaa gtcggaatcg ctagtaatcg 1320
cggatcagca cgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1380
cgagagtttg taacacccga agtcggtgag gtaacctttg gagccagccg cctaaggtca 1440
agatgtt 1447
Claims (8)
1. A streptococcus salivarius QS-20 with antioxidant activity, wherein the streptococcus salivarius QS-20 is classified asStreptococcus salivariusThe strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC 7.456.
2. The streptococcus salivarius QS-20 having an antioxidant activity according to claim 1, wherein the streptococcus salivarius QS-20 is white raised colonies with a smooth surface, colony diameters of 0.2-1mm, cells are spherical or oval, and have a diameter of not more than 1 μm and are arranged in a chain.
3. The streptococcus salivarius QS-20 having an antioxidant activity according to claim 1, wherein the 16S rDNA nucleotide sequence of said streptococcus salivarius QS-20 is shown in SEQ ID No. 1.
4. Use of streptococcus salivarius QS-20 according to any of claims 1-3 for the preparation of an antioxidant, wherein said streptococcus salivarius QS-20 is antioxidative by scavenging DPPH radicals.
5. The use of streptococcus salivarius QS-20 according to claim 4 for the preparation of an antioxidant, wherein the antioxidant is active for a period of time ranging from 12h to 36h.
6. Use of streptococcus salivarius QS-20 according to any of claims 1 to 3 for the preparation of an acidogenic acid tolerant starter culture.
7. The use of streptococcus salivarius QS-20 according to claim 6 for the preparation of a starter culture, wherein the starter culture has a fermentation time of 0-3h.
8. Use of streptococcus salivarius QS-20 according to any of claims 1 to 3 for the preparation of a bacteriostatic agent capable of inhibiting escherichia coli, bacillus subtilis, staphylococcus aureus, salmonella and pseudomonas aeruginosa.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH054927A (en) * | 1991-06-28 | 1993-01-14 | Aizo Matsushiro | Lactic acid-containing composition and production thereof |
| BRPI0605568A (en) * | 2006-12-06 | 2008-07-22 | Vera Fantinato | composition, food product, fermented milk, fermentation milk preparation process and use of streptococcus salivarius subsp salivarius bacteria |
| CN101538545B (en) * | 2009-04-08 | 2011-07-20 | 任发政 | Lactobacillus salivarius and food composition thereof |
| RU2567148C1 (en) * | 2014-12-24 | 2015-11-10 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Горский государственный аграрный университет" | Streptococcus salivarius VKPM V-11177 STRAIN USED FOR PRODUCTION OF FERMENTED MILK PRODUCTS AND PRO-BIOTIC PREPARATIONS |
| KR102173646B1 (en) * | 2018-06-28 | 2020-11-03 | 주식회사 세바바이오텍 | Streptococcus salivarius sbk4 strain isolated from skin and functional cosmetic composition comprising Streptococcus salivarius sbk4 strain |
| JP6651202B1 (en) * | 2018-10-17 | 2020-02-19 | 株式会社北岡本店 | A novel lactic acid bacterium belonging to Streptococcus salivarius and its use |
| RU2698032C1 (en) * | 2018-11-08 | 2019-08-21 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Горский государственный аграрный университет" | Strain of lactobacilli streptococcus salivarius - producer of lactic acid and antibiotic substances |
| JP2023540973A (en) * | 2020-09-04 | 2023-09-27 | ユニバーシタット アントウェルペン | New probiotic Streptococcus salivarius strains and their uses |
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