CN114480167A - Lactococcus lactis MD-622 and application thereof - Google Patents

Lactococcus lactis MD-622 and application thereof Download PDF

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CN114480167A
CN114480167A CN202111586675.5A CN202111586675A CN114480167A CN 114480167 A CN114480167 A CN 114480167A CN 202111586675 A CN202111586675 A CN 202111586675A CN 114480167 A CN114480167 A CN 114480167A
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lactococcus lactis
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刘庆军
沈鹤霄
张帆
李国龙
刘慧敏
彭文聪
熊云
王峰
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Maintain Biomedical Wuhan Co ltd
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Abstract

The invention provides lactobacillus lactis MD-622 and application thereof. The Lactobacillus lactis MD-622 can promote the production of protease, amylase and cellulase, and has potential capability of promoting the digestion of human intestinal tract. In addition, the lactobacillus lactis MD-622 can promote the growth and development of embryonic stem cells to a certain extent.

Description

Lactococcus lactis MD-622 and application thereof
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactococcus lactis MD-622 and application thereof.
Background
Lactococcus lactis (l.lactis) is a prokaryotic microorganism belonging to the phylum firmicutes, class bacilli, order lactobacillales, family streptococcaceae, genus Lactococcus, and is an important model bacterium in the genus Lactococcus.
Lactis are widely present in dairy and plant products, widely used in the food industry, are not pathogenic to humans and animals, and are food grade microorganisms (GRAS) that are generally recognized as safe. However, there are many actual species of lactococcus lactis, and many lactococcus lactis and their effects have not been known and utilized.
Disclosure of Invention
Based on the above, there is a need for lactococcus lactis MD-622 and applications thereof, wherein lactococcus lactis MD-622 can promote in vivo production of protease, amylase and cellulase, improve intestinal discomfort, and promote embryonic stem cell growth and development to a certain extent.
The invention adopts the following technical scheme:
the invention provides lactococcus lactis MD-622 which is preserved in China general microbiological culture Collection center (CGMCC) at 11 months and 15 days 2021, wherein the preservation number is CGMCC No.23902, and the preservation unit address is as follows: beijing in China.
The sequence of the 16S rRNA of the lactococcus lactis is shown in SEQ ID NO. 1.
In some of these embodiments, the culture conditions of lactococcus lactis are: MRS culture medium, pH6.2-6.6.
In some of these embodiments, the lactococcus lactis is capable of producing a protease, an amylase, a cellulase.
The invention also provides a composition containing the lactococcus lactis MD-622 and secretion products or metabolites thereof.
The invention also provides application of the lactococcus lactis MD-622 in preparation of probiotic products. The probiotic product is a medicine, functional food or health-care product for improving intestinal diseases, or the probiotic product is a medicine, functional food or health-care product for promoting the growth and development of embryonic stem cells.
The invention also provides a probiotic preparation which comprises the lactococcus lactis MD-622 and auxiliary materials.
The invention also provides a probiotic preparation which comprises the secretion product of lactococcus lactis MD-622.
The invention has the beneficial effects that:
compared with the prior art, the lactococcus lactis MD-622 is obtained by screening a large number of research tests, and the lactococcus lactis MD-622 can promote in vivo production of protease, amylase and cellulase, improve intestinal health and promote growth and development of embryonic stem cells to a certain extent.
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FIG. 1 is a photograph of a colony of lactococcus lactis MD-622.
FIG. 2 is an enzyme production test chart of lactococcus lactis MD-622.
Detailed Description
The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention.
The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by a person skilled in the art based on the specific embodiments of the present invention without any inventive step are within the scope of the present invention.
In the examples of the present invention, all the raw material components are commercially available products well known to those skilled in the art, unless otherwise specified; in the examples of the present invention, unless otherwise specified, all technical means used are conventional means well known to those skilled in the art.
The key reagent composition is as follows:
MRS culture medium: 10g/L of peptone, 5g/L of beef extract powder, 4g/L of yeast extract powder, 20g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of triammonium citrate, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.05g/L of manganese sulfate, 15g/L of agar, 801.0 g/L of tween and 6.2 +/-0.2 of pH value.
Example 1 screening of lactic acid bacteria
Taking feces of healthy adults about 25 years old, diluting with distilled water, coating the feces in an MRS (lactic acid bacteria culture medium), carrying out inverted culture at 37 ℃, selecting single colonies with different forms on the culture medium, streaking, continuing to carry out purification culture, and separating strains with the numbers of 5b4M2, cb18M1, PS128, XS10M8, XS14M1 and XS14M 5.
EXAMPLE 2 enzyme production Activity test
The enzyme production activity tests were carried out on strains 5b4M2, cb18M1, PS128, XS10M8, XS14M1 and XS14M5, respectively:
(1) the strains are respectively inoculated into a protease screening culture medium containing 10g/L of skimmed milk powder, 10g/L of peptone and 15g/L of agar powder, and cultured at 37 ℃. Further, the production of protease in the culture broth was examined by the protease hydrolysis loop method.
The statistical results are shown in the following table:
statistics table of protease production of different bacteria (N ═ 3)
Figure BDA0003428012440000031
Figure BDA0003428012440000041
(2) Inoculating the above strains with a solution containing 13mM Tris-HCl, 75mM NaCl, and 1.3mM CaCl2(pH8.0), agar 15g/L, 10mg/L rhodamine B, and polyethylene olive oil emulsion 31.25mL, and culturing at 37 deg.C. Further detecting the lipase production condition in the culture solution by using a lipase transparent ring method.
The statistical results are shown in the following table:
statistical table of lipase production of different bacteria (N ═ 3)
Figure BDA0003428012440000042
Remarking: "-" represents no transparent circles.
(3) Inoculating the above strains into amylase screening culture medium containing 5g/L yeast extract, 15g/L soluble starch, and 15g/L agar powder, respectively, and culturing at 37 deg.C. And further detecting the amylase production condition in the culture solution by adopting an amylase hydrolysis loop method.
The statistical results are shown in the following table:
statistics table for amylase production by different bacteria (N ═ 3)
Figure BDA0003428012440000043
Figure BDA0003428012440000051
Remarking: "-" represents none and "+" represents presence.
(4) The strains are respectively inoculated into cellulase screening culture media containing 5g/L of yeast extract, 5g/L of carboxymethyl cellulose and 15g/L of agar powder, and cultured at 37 ℃. And further detecting the cellulase production condition in the culture solution by adopting a cellulase hydrolysis loop method.
The statistical results are shown in the following table:
statistical table of cellulase production of different bacteria (N ═ 3)
Figure BDA0003428012440000052
Remarking: "-" represents none, and "+" represents presence.
The detection proves that only the cb18m1 strain can simultaneously produce protease, amylase and cellulase. As shown in detail in fig. 2.
The cb18m1 strain is checked and identified, and the 16s sequence of the cb18m1 strain is tested to be 5_ TSS 20211101-027-00230:
ggggcgtgtgctatgatgcagttgagcgctgaaggttggtacttgtaccgactggatgagcagcgaacgggt gagtaacgcgtggggaatctgcctttgagcgggggacaacatttggaaacgaatgctaataccgcataaaaacttta aacacaagttttaagtttgaaagatgcaattgcatcactcaaagatgatcccgcgttgtattagctagttggtgaggtaa aggctcaccaaggcgatgatacatagccgacctgagagggtgatcggccacattgggactgagacacggcccaa actcctacgggaggcagcagtagggaatcttcggcaatggacgaaagtctgaccgagcaacgccgcgtgagtga agaaggttttcggatcgtaaaactctgttggtagagaagaacgttggtgagagtggaaagctcatcaagtgacggta actacccagaaagggacggctaactacgtgccagcagccgcggtaatacgtaggtcccgagcgttgtccggattta ttgggcgtaaagcgagcgcaggtggtttattaagtctggtgtaaaaggcagtggctcaaccattgtatgcattggaaa ctggtagacttgagtgcaggagaggagagtggaattccatgtgtagcggtgaaatgcgtagatatatggaggaaca ccggtggcgaaagcggctctctggcctgtaactgacactgaggctcgaaagcgtggggagcaaacaggattagat accctggtagtccacgccgtaaacgatgagtgctagatgtagggagctataagttctctgtatcgcagctaacgcaat aagcactccgcctggggagtacgaccgcaaggttgaaactcaaaggaattgacgggggcccgcacaagcggtg gagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatactcgtgctattcctagagatagga agttccttcgggacacgggatacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgc aacgagcgcaacccctattgttagttgccatcattaagttgggcactctaacgagactgccggtgataaaccggagg aaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggatggtacaacgag tcgcgagacagtgatgtttagctaatctcttaaaaccattctcagttcggattgtaggctgcaactcgcctacatgaagt cggaatcgctagtaatcgcggatcagcacgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacac cacgggagttgggagtacccgaagtaggttgcctaaccgcaaggagggcgctcctaagtagaccgattg(SEQ IN NO.1)。
the cb18m1 strain was named Lactococcus lactis (Lactococcus lactis) MD-622 in combination with colony morphology and 16S sequence.
Lactococcus lactis (Lactococcus lactis) MD-622 was collected by the China general microbiological culture Collection center (CGMCC) at 11/15/2021, and culture conditions and survival conditions of the culture were as follows: all are cultured in lactic acid bacteria culture Medium (MRS) with pH of 6.2-6.6, and kept standing overnight in a constant temperature incubator at 37 ℃. Ultra-low temperature freezing for long-term storage is recommended.
Example 3 virulence experiments and safety testing
The cb18m1 strain is inspected, and a toxicological test and a safety test are carried out, and the results show that: the culture of the cb18m1 strain does not endanger human health or animal and plant pathogens or pollute the environment.
Example 4
Culturing bone cells (C3H10T1/2) by culturing C3H1OT1/2 cell line at 37 deg.C and 5% CO2The culture was performed in a DMEM high-sugar medium containing 10% fetal bovine serum, 100U/mL penicillin and 100. mu.g/mL streptomycin.
Continuously activating to-be-detected inactivated lactococcus lactis strain with MRS liquid culture medium for 3 generations, washing bacteria with sterile PBS buffer solution, and adjusting density to 3 × 108And (mL). Inoculating 2% volume of MRS liquid culture medium, shaking, sealing, and fermenting in 37 deg.C gas bath constant temperature oscillator; after 1 week, the fermentation broth was centrifuged at 4000 r/min for 30min, and the supernatant was filtered off with a 0.2 μm microfiltration membrane filter.
Experimental groups: the osteoblasts C3H10T1/2 were stimulated with supernatant from Lactococcus lactis (Lactococcus lactis) MD-622 to be inactivated.
The blank group was stimulated with the same volume of 2.0% formaldehyde as the experimental group.
The MTT method is used for detecting the growth condition of the cells, and the method comprises the following steps: the C3H1OT1/2 cells in logarithmic growth phase are randomly divided into a test group and a control group which are 5 multiplied by 105The cells were seeded in 96-well plates at a density of one mL/mL, 100. mu.L of RPMI-4640 cell culture (containing 10% fetal bovine serum) was added to each well, and the mixture was incubated at 37 ℃ with 5% CO2Culturing for 24h under the condition, and removing supernatant.
The experimental group was added with 200. mu.L of fermentation broth, the volume ratio of fermentation broth to cell culture fluid was 1: 4.
And adding 200 mu L of PBS buffer solution (the volume ratio of the PBS buffer solution to the cell culture solution is the same as that of the cell culture solution), continuously culturing for 72h, adding 10 mu L of tetrazolium blue MTT solution into each well, washing and removing supernatant after 4h, adding 150 mu L of dimethyl sulfoxide into each group of at least 6 parallel wells, and measuring the Optical Density (OD) value of each group at the wavelength of 570nm by using enzyme labeling.
The conversion, results are statistically shown in the following table:
statistical table of growth rate of osteocyte C3H10T1/2
Numbering Name (R) Bone cell C3H10T1/2 growth Rate (%)
CK Cells 100.00％
Blank space Ctrl 111.43％
cb18m1 Lactobacillus lactis 81.72％
As can be seen from the above table, the cb18m1 Lactobacillus lactis has an inhibitory effect on the growth of osteocytes C3H10T 1/2.
Example 5
Culturing mouse embryonic stem cells CE 3: embryonic stem cells were cultured in a culture medium of 90% DMEM (high glucose), 10% Fetal Bovine Serum (FBS), 1% penicillin, 1% streptomycin.
Continuously activating to-be-detected inactivated lactococcus lactis strain with MRS liquid culture medium for 3 generations, washing bacteria with sterile PBS buffer solution, and adjusting density to 3 × 108And (mL). Inoculating MRS liquid culture medium with 2% volume, and shakingHomogenizing, sealing, and fermenting in 37 deg.C gas bath constant temperature oscillator; after 1 week, the fermentation broth was centrifuged at 4000 r/min for 30min, and the supernatant was filtered off with a 0.2 μm microfiltration membrane filter.
The method for detecting the growth of the embryonic cells by using the MTT method through stimulating the supernatant of the inactivated strain Lactococcus lactis (Lactococcus lactis) MD-622 to be detected comprises the following steps:
the CE3 cells in logarithmic growth phase are randomly divided into a test group and a control group according to the ratio of 5 multiplied by 105The cells were seeded in 96-well plates at a density of one mL/mL, 100. mu.L of RPMI-4640 cell culture (containing 10% fetal bovine serum) was added to each well, and the mixture was incubated at 37 ℃ with 5% CO2Culturing under the condition for 24h, and removing supernatant.
The experimental group was added with 200. mu.L of fermentation broth, the volume ratio of fermentation broth to cell culture fluid was 1: 4.
And adding 200 mu L of PBS buffer solution (the volume ratio of the PBS buffer solution to the cell culture solution is the same as that of the cell culture solution), continuously culturing for 72h, adding 10 mu L of tetrazolium blue MTT solution into each well, washing and removing supernatant after 4h, adding 150 mu L of dimethyl sulfoxide into each group of at least 6 parallel wells, and measuring the Optical Density (OD) value of each group at the wavelength of 570nm by using enzyme labeling.
The conversion, results are statistically shown in the following table:
statistical table of growth rate of CE3 embryonic stem cells
Numbering Name (R) Growth rate (%) of embryonic Stem cell CE3
CK Cells 100.00％
Blank space Ctrl 95.73％
cb18m1 Lactobacillus lactis 126.54％
As can be seen from the above table, the cb18m1 Lactobacillus lactis has a promoting effect on the growth and development of the embryonic stem cell CE 3.
Example 6
The embodiment provides a probiotic preparation containing Lactococcus lactis (Lactococcus lactis) MD-622, which comprises Lactococcus lactis MD-622 and preparation auxiliary materials.
In conclusion, the lactococcus lactis MD-622 is obtained by screening a large number of research tests, and the lactococcus lactis MD-622 can promote in vivo production of protease, amylase and cellulase, improve intestinal health and promote growth and development of embryonic stem cells to a certain extent.
It should be noted that the above examples are only for further illustration and description of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment, and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Meiyi Adoration biomedical (Wuhan) Co., Ltd
<120> Lactobacillus lactis MD-622 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1445
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggggcgtgtg ctatgatgca gttgagcgct gaaggttggt acttgtaccg actggatgag 60
cagcgaacgg gtgagtaacg cgtggggaat ctgcctttga gcgggggaca acatttggaa 120
acgaatgcta ataccgcata aaaactttaa acacaagttt taagtttgaa agatgcaatt 180
gcatcactca aagatgatcc cgcgttgtat tagctagttg gtgaggtaaa ggctcaccaa 240
ggcgatgata catagccgac ctgagagggt gatcggccac attgggactg agacacggcc 300
caaactccta cgggaggcag cagtagggaa tcttcggcaa tggacgaaag tctgaccgag 360
caacgccgcg tgagtgaaga aggttttcgg atcgtaaaac tctgttggta gagaagaacg 420
ttggtgagag tggaaagctc atcaagtgac ggtaactacc cagaaaggga cggctaacta 480
cgtgccagca gccgcggtaa tacgtaggtc ccgagcgttg tccggattta ttgggcgtaa 540
agcgagcgca ggtggtttat taagtctggt gtaaaaggca gtggctcaac cattgtatgc 600
attggaaact ggtagacttg agtgcaggag aggagagtgg aattccatgt gtagcggtga 660
aatgcgtaga tatatggagg aacaccggtg gcgaaagcgg ctctctggcc tgtaactgac 720
actgaggctc gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgccgta 780
aacgatgagt gctagatgta gggagctata agttctctgt atcgcagcta acgcaataag 840
cactccgcct ggggagtacg accgcaaggt tgaaactcaa aggaattgac gggggcccgc 900
acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga 960
catactcgtg ctattcctag agataggaag ttccttcggg acacgggata caggtggtgc 1020
atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1080
ctattgttag ttgccatcat taagttgggc actctaacga gactgccggt gataaaccgg 1140
aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct 1200
acaatggatg gtacaacgag tcgcgagaca gtgatgttta gctaatctct taaaaccatt 1260
ctcagttcgg attgtaggct gcaactcgcc tacatgaagt cggaatcgct agtaatcgcg 1320
gatcagcacg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg 1380
ggagttggga gtacccgaag taggttgcct aaccgcaagg agggcgctcc taagtagacc 1440
gattg 1445

Claims (9)

1. Lactococcus lactis MD-622 has been deposited in China general microbiological culture Collection center (CGMCC) at 11 months and 15 days 2021 with the deposition number of CGMCC No. 23902.
2. Lactococcus lactis MD-622 according to claim 1, wherein the lactococcus lactis has the 16S rRNA sequence shown in SEQ ID No. 1.
3. Lactococcus lactis MD-622 according to claim 1, wherein said lactococcus lactis is cultured under conditions selected from the group consisting of: MRS culture medium, pH6.2-6.6.
4. Lactococcus lactis MD-622 according to claim 1, wherein said lactococcus lactis is capable of producing a protease, an amylase, a cellulase.
5. A composition comprising lactococcus lactis MD-622 as set forth in any one of claims 1 to 4.
6. Use of lactococcus lactis MD-622 and its secretory products or metabolites according to any one of claims 1 to 4 in the preparation of probiotic products.
7. The use according to claim 6, wherein the probiotic product is a drug, functional food or health product for improving intestinal diseases, or a drug, functional food or health product for promoting the growth and development of embryonic stem cells.
8. A probiotic, characterized by comprising lactococcus lactis MD-622 according to any one of claims 1 to 4, the composition according to claim 5.
9. The probiotic according to claim 8, characterized in that it further comprises an adjuvant.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115181695A (en) * 2022-06-27 2022-10-14 广东粤港澳大湾区国家纳米科技创新研究院 Lactobacillus plantarum5b4m2 and application thereof
CN117660246A (en) * 2023-12-06 2024-03-08 河北一然生物科技股份有限公司 Lactococcus lactis subspecies S28 for high-yield metabolic enzyme and application thereof

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