CN115093989A - 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
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- A61K35/74—Bacteria
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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Abstract
The invention discloses a streptococcus salivarius QS-20 with antioxidant activity and application thereof. The classification name of the streptococcus salivarius QS-20 isStreptococcus salivariusThe strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC 7.456, and can effectively remove oxidation substances such as DPPH free radicals, hydrogen peroxide and the like; also has good acid-producing and acid-resisting properties and bile salt-resisting capability, is favorable for the permanent planting of the bacteria in intestinal tracts, andit has excellent inhibitory activity against various pathogenic bacteria such as Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Salmonella and Pseudomonas aeruginosa. Therefore, QS-20 of streptococcus salivarius has wide application scenes.
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 initiation of the neonatal gut immune system, the development of the infant gut immune function and the programmed process, but also in protecting the infant from diseases and infections during the growth process. At present, microorganisms which have been isolated and identified from breast milk are mainly staphylococcus, streptococcus, enterococcus, lactobacillus, bifidobacterium, leuconostoc and the like. Wherein, staphylococcus, streptococcus, bifidobacterium and lactobacillus are the dominant flora in the breast milk and have important probiotic function. Therefore, the research and development of the human milk streptococcus have important significance.
Disclosure of Invention
The invention aims to provide a streptococcus salivarius QS-20 with antioxidant activity and application thereof, wherein the streptococcus salivarius QS-20 has antioxidant activity, acid production characteristic, bile salt resistance and bacteriostatic ability.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides streptococcus salivarius QS-20 with antioxidant activity, and the classification of the streptococcus salivarius QS-20 isStreptococcus salivariusThe culture medium is preserved in China general microbiological culture Collection center, and the address is as follows: xilu No.1 Hospital No. 3, Chaojing, Chaoyang, with a preservation number of CGMCC 7.456, and a preservation date: 13 months 1 in 2022; the result of the viability test of the biomaterial is as follows: and (6) survival.
Furthermore, the streptococcus salivarius QS-20 is a white protruding colony, the surface is smooth, the diameter of the colony is 0.2-1mm, cells are spherical or oval, the diameter is not more than 1 mu m, and the cells are arranged in a chain shape.
Furthermore, the streptococcus salivarius QS-20 has antioxidant activity, acid production property, bile salt resistance and bacteriostatic ability.
Further, the nucleotide sequence of QS-20 of streptococcus salivarius is shown as SEQ ID No.1
The invention also provides application of QS-20 in streptococcus salivarius in preparing an antioxidant.
Further, the action time of the antioxidant is 12-36 h.
Furthermore, the streptococcus salivarius QS-20 achieves the effect of resisting oxidation by eliminating DPPH free radicals and hydrogen peroxide.
The invention also provides application of the streptococcus salivarius QS-20 in preparation of a leavening agent.
Further, the fermentation time of the leavening agent is 0-3 h.
The invention also provides application of the streptococcus salivarius QS-20 in preparation of a bacteriostatic agent.
Further, the bacteriostatic agent 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 probiotic is separated from the breast milk, and the streptococcus salivarius QS-20 with good oxidation resistance is obtained by taking DPPH clearance as a screening index, has good oxidation resistance activity, and can effectively remove oxidation substances such as DPPH free radicals and hydrogen peroxide; also has good acid-producing and acid-resisting properties, and provides basis for the application of the compound in fermented foods. In addition, the streptococcus salivarius QS-20 also has bile salt resistance, is beneficial to colonization 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, QS-20 of streptococcus salivarius has wide application scenes.
Drawings
FIG. 1 is an electrophoretogram of 16S rDNA amplification PCR products of a part of strains.
FIG. 2 is a QS-20 strain phylogenetic tree analysis.
FIG. 3 is a colony morphology and a cell morphology map of QS-20 of Streptococcus salivarius.
FIG. 4 shows the growth and acidogenesis curves of QS-20 of Streptococcus salivarius.
FIG. 5 is a graph showing DPPH free radical scavenging effect of QS-20 in Streptococcus salivarius.
FIG. 6 is an acid resistance curve for Streptococcus salivarius QS-20.
FIG. 7 is a bile salt resistance curve for S.salivarius QS-20.
FIG. 8 is a graph showing the inhibitory effect of QS-20 of Streptococcus salivarius on bacteria.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the following specific examples.
In the following examples, unless otherwise specified, the experimental methods used were all conventional methods, and materials, reagents and the like used were all available from biological or chemical reagents companies.
Example 1: isolation screening and characterization of QS-20 of Streptococcus salivarius
1. Isolation and identification
Fresh breast milk is diluted by physiological saline according to different concentrations and coated on MRS solid culture medium, and is subjected to inverted culture at 37 ℃ for 48 h. Selecting a single bacterial colony with smooth and white or milk white thallus for gram staining and microscopic examination, selecting rod-shaped and gram-positive bacteria, and streaking and purifying for 2-3 times on an MRS solid culture medium. And selecting a streaked and purified single colony, inoculating the single colony to an MRS liquid culture medium, continuously culturing for 24-48 h at 37 ℃, mixing bacterial liquid and sterilized glycerol according to a ratio of 1: 1, and storing in a refrigerator at-80 ℃ for later use.
(1) Extracting genome DNA by adopting a radix asparagi genome kit;
(2) 16S rDNA PCR amplification, adopting universal primer,
the upstream primer 27F: GAGAGTTTGATCCTGGCTCAG, and the adhesive tape is used for adhering the film to a substrate,
the downstream primer 1492R: TACGGCTACCTTGTTACGAC are provided.
The PCR reaction system is 50 ul: taq Reaction Buffer (10X) 5ul, PCR Dye 5ul, dNTPs 1ul, Taq Dymerase 0.5ul, DNA template 2ul, 27F 2ul, 1492R 2ul, water to make up to 50 ul.
The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5 min; circulating for 30 times (denaturation at 95 deg.C for 30 s; annealing at 55 deg.C for 30 s; extension at 72 deg.C for 90 s); extension at 72 ℃ for 7min, and extension at the end for 20 ℃ for 3 min.
(3) Sequencing of PCR amplification products
The PCR product was detected by 1% agarose gel electrophoresis and sent to Qingdao Biotech for sequencing. The sequences were determined to be aligned for homology with the GenBank database by the BLAST program.
16 bacteria are separated from three different breast milk samples by a transparent circle method, 16S rDNA amplification is carried out to obtain target bands (figure 1) of about 1500 bp, and the nucleotide sequences are shown as SEQ ID No. 1.
Sequencing the target band and analyzing phylogenetic tree, and obtaining QS-20 strain and QS-20 strain from the phylogenetic tree shown in FIG. 2Streptococcus salivariusThe relationship is recent and therefore it belongs to the streptococcus salivarius.
Strain QS-20 is deposited, the unit of deposit of streptococcus salivarius QS-20: china general Committee for culture Collection of microorganismsMicrobial center (CGMCC); address: west road No.1, north zhou zhao yang district, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 1 month and 13 days 2022; the classification is named as: salivary streptococcusStreptococcus salivarius,The preservation number is CGMCC 7.456, and the detection result of the viability of the biological material is as follows: and (6) survival.
2. Morphological observations
The QS-20 strain was observed for colony morphology and gram-stained cell morphology, as shown in FIG. 3. QS-20 strain white convex colony, smooth surface, 0.2-1mm diameter, spherical or oval cell with diameter not more than 1 μm, chain arrangement, and precipitation growth in liquid culture medium. The QS-20 strain can produce acid to form a transparent ring on an MRS culture medium added with calcium carbonate, the gram staining result shows that the QS-20 strain is gram positive bacteria, meanwhile, the catalase test of the strain is negative, and the result further shows that the QS-20 strain is streptococcus in the gram positive bacteria, which is consistent with the result of the phylogenetic tree analysis.
Example 2: biochemical Properties of Streptococcus salivarius QS-20
1. Growth and acid production curves of the QS-20 Strain of Streptococcus salivarius
Referring to fig. 4, the QS-20 strain can rapidly enter the logarithmic growth phase within a short time, has strong adaptability and short adaptation period, enters the exponential growth phase within 5-15h and then enters the stationary phase, and has no obvious decay phase within 52 h. In the exponential growth phase, the acid-producing property of streptococcus salivarius QS-20 is strong, and the pH value is obviously reduced, so that the streptococcus salivarius QS-20 is suitable for serving as a leavening agent.
2. Determination of antioxidant Activity of Streptococcus salivarius QS-20 Strain
The DPPH free radical is reduced by the antioxidant and becomes stable DPPH molecule, the color is changed from dark purple to light yellow, and the purple intensity of DPPH is inversely proportional to the antioxidant activity of the sample. DPPH has a stable structure, and if DPPH can be eliminated, the test strain has the chain reaction effects of reducing hydrogen peroxide and eliminating various free radicals. The QS-20 strain of streptococcus salivarius is inoculated in MRS broth liquid culture medium, and the antioxidant activity of fermentation supernatant is measured at different culture time.
As shown in FIG. 5, it can be seen that QS-20 strain has high DPPH clearance rate, which reaches more than 70% after 12h and more than 80% after 36h, and shows strong antioxidant activity.
3. Determination of acid resistance of QS-20 Strain of Streptococcus salivarius
QS-20 strain is inoculated into MRS broth liquid culture medium with pH 3.0 for culturing for 3 and 5 hours, the liquid culture medium without thalli and with pH 3.0 is used as a blank, OD600nm is measured, the culture time is used as abscissa, the absorbance value is used as slave coordinate, and a broken line trend graph is drawn.
As can be seen in FIG. 6, the absorbance gradually increased within 3h, indicating that QS-20 strain can survive and grow; the absorbance at 5h was not changed much compared to 3h, indicating that the growth of the strain was inhibited. Therefore, the maximum survival tolerance time of the strain under the acidic condition is 0-3 h.
4. Determination of bile salt resistance of QS-20 strain of streptococcus salivarius
QS-20 strain is inoculated into MRS broth liquid culture medium containing different concentrations of bile salt (0.1%, 0.2%, 0.3%) and cultured for 3 and 5 hours, the liquid culture medium containing no thalli and different concentrations of bile salt is used as a blank, the absorbance of OD600nm is measured, the culture time is used as an abscissa, the absorbance value is used as an ordinate, and a broken line trend graph is drawn.
As shown in FIG. 7, QS-20 of streptococcus salivarius can survive and grow within 5h, and after 3h, the high concentration of bile salt can inhibit the growth of the strain to some extent.
Example 3: analysis of bacteriostatic Capacity of Streptococcus salivarius QS-20
Culturing Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Salmonella and Pseudomonas aeruginosa, and detecting the antibacterial activity of Streptococcus salivarius QS-20 on the strains.
The results in FIG. 8 show that the QS-20 strains of Streptococcus salivarius have inhibitory activity against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Salmonella, and Pseudomonas aeruginosa, among which the inhibitory activity against Bacillus subtilis and Pseudomonas aeruginosa is stronger.
The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described in the foregoing embodiments, or equivalents may be substituted for some of the features thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
<110> Qingdao agricultural university
<120> streptococcus salivarius QS-20 with antioxidant activity and application thereof
<141> 2022-04-08
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<170> SIPOSequenceListing 1.0
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aggcgacgat acatagccga cctgagaggg tgatcggcca cactgggact gagacacggc 300
ccagactcct acgggaggca gcagtaggga atcttcggca atgggggcaa ccctgaccga 360
gcaacgccgc gtgagtgaag aaggttttcg gatcgtaaag ctctgttgta agtcaagaac 420
gagtgtgaga gtggaaagtt cacactgtga cggtagctta ccagaaaggg acggctaact 480
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cgctgaggct cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt 780
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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 (10)
1. A strain of streptococcus salivarius QS-20 with antioxidant activity, wherein the classification of the streptococcus salivarius QS-20 is shown asStreptococcus salivariusThe strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC 7.456.
2. The process according to claim 1, wherein said QS-20 streptococcus salivarius are white protruding colonies with smooth surface, 0.2-1mm diameter colonies, spherical or oval cells with diameter not more than 1 μm, and chain-like arrangement.
3. The method according to claim 1, wherein the nucleotide sequence of QS-20 is SEQ ID No. 1.
4. Use of a streptococcus salivarius QS-20 as claimed in any of claims 1 to 3 in the preparation of an antioxidant.
5. The use of QS-20 in the preparation of an antioxidant according to claim 4, wherein the antioxidant has an action time of 12-36 hours.
6. Use of QS-20 in the preparation of an antioxidant according to claim 4, wherein the QS-20 has antioxidant activity by scavenging DPPH free radicals and hydrogen peroxide.
7. Use of a streptococcus salivarius QS-20 as claimed in any of claims 1 to 3 in the preparation of a starter culture.
8. The use of QS-20 in accordance with claim 6, for the preparation of a starter culture, wherein the starter culture has a fermentation time of 0-3 h.
9. Use of a streptococcus salivarius QS-20 as claimed in any of claims 1 to 3 in the manufacture of a bacteriostatic agent.
10. Use of QS-20 to streptococcus salivarius for the preparation of a bacteriostatic agent according to claim 9 wherein the bacteriostatic agent is capable of inhibiting escherichia coli, bacillus subtilis, staphylococcus aureus, salmonella and pseudomonas aeruginosa.
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Cited By (1)
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