CN117645965B - Lactobacillus casei rhamnosus with intestinal indole degrading and chronic renal failure relieving functions - Google Patents

Lactobacillus casei rhamnosus with intestinal indole degrading and chronic renal failure relieving functions Download PDF

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CN117645965B
CN117645965B CN202410117188.1A CN202410117188A CN117645965B CN 117645965 B CN117645965 B CN 117645965B CN 202410117188 A CN202410117188 A CN 202410117188A CN 117645965 B CN117645965 B CN 117645965B
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lactobacillus rhamnosus
indole
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intestinal
lactobacillus
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CN117645965A (en
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王倩
段治
郭超群
崔洪昌
程淑敏
李鑫萍
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Qingdao Vland Biotech Group Co Ltd
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Abstract

The invention provides a lactobacillus rhamnosus strain with functions of degrading intestinal indole and relieving chronic renal failure, belonging to the technical field of screening and application of probiotics. The preservation number of the lactobacillus rhamnosus is CCTCC NO: M20221668. The lactobacillus rhamnosus is separated from yoghurt and has strong degradation capability on tryptophan metabolite indole in intestinal tracts. The lactobacillus rhamnosus has strong tolerance to artificial intestinal gastric juice, is sensitive to common antibiotics such as erythromycin and ampicillin, does not generate hemolysin, can not dissolve blood cells, and has good biological safety. The lactobacillus rhamnosus screened by the invention can be used singly or in combination with various probiotics, is used for maintaining human health and has wide application prospect.

Description

Lactobacillus casei rhamnosus with intestinal indole degrading and chronic renal failure relieving functions
Technical Field
The invention belongs to the technical field of screening and application of probiotics, and particularly relates to lactobacillus rhamnosus with functions of degrading intestinal indole and relieving chronic renal failure.
Background
Chronic Kidney Disease (CKD) is a type of kidney disease in which the renal structure or function is abnormal for a variety of reasons, not less than 3 months, and the renal function is slowly depleted over several months or even years. As the course of the disease progresses, CKD eventually evolves into end stage renal disease. CKD has high incidence and high mortality, but has low awareness and low control rate, and in recent years, the incidence of chronic kidney disease on the global scale has gradually increased, and has become a global disease which seriously threatens the health of people after heart disease, diabetes and tumor. To date, chronic renal failure has lacked an effective treatment means mainly by hemodialysis and kidney transplantation, but hemodialysis requires lifelong dialysis of patients, which IS costly, and protein-bound uremic toxins such as Indoxyl Sulfate (IS) and p-cresol sulfate (PCS) are extremely difficult to remove by hemodialysis, and kidney transplantation also faces the dilemma of shortage of kidney sources and higher cost.
Numerous researchers have used prebiotics, probiotic dietary supplements as potential strategies for improving changes in the microbiota associated with CKD and its complications. Ingestion of a certain amount of lactic acid bacteria from the digestive tract not only inhibits pathogenic bacteria from growing to reduce the production of the above toxins, but also breaks down certain uremic toxins. Smienho and the like are used for treating end-stage renal failure by freeze-dried lactobacillus, and found that the content of dimethylamine and methylene nitrosamine in the body can be reduced, the content of serum albumin can be increased, the quality of the body is increased, and the appetite is improved; the bifidobacterium preparation is used for treating chronic renal failure patients in China Song Shangming and the like, and can reduce blood urea nitrogen, but the creatinine is not obviously reduced. Research results show that at the end of diet intervention, the intervention group containing bifidobacterium longum BL-G301 has obviously reduced urea level in blood, increased albumin level in blood and reduced phosphate level, so that the safety of the intestinal physiological bacteria can be obviously improved by selecting the intestinal physiological bacteria. Aiming at the formation mechanism of protein-binding toxin indoxyl sulfate, the development of functional probiotics capable of degrading indole in intestinal tracts, reducing the level of urotoxin indoxyl sulfate in patients and improving the life quality of patients with chronic renal failure is a target of effort at present.
Disclosure of Invention
The invention aims to provide a novel lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) and application thereof. The rhamnose cheese bacillus is separated from yoghurt, has strong degradation capability on tryptophan metabolite indole in intestinal tracts, wherein the indole is a precursor substance of protein-bound urotoxin indoxyl sulfate, and the content of the indole is reduced, so that the indoxyl sulfate level in a patient is reduced, the symptoms of the patient suffering from renal failure are relieved, and the rhamnose cheese bacillus can be applied to the adjuvant therapy of the patient suffering from renal failure, prevent more complications and prolong the service life of the patient.
The lactobacillus rhamnosus provided by the invention is VHProbi V6156 (Lacticaseibacillus rhamnosus VHProbi V6156), and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20221668 in 10-month 26 of 2022.
The 16s rDNA sequence of the lactobacillus rhamnosus VHProbi V6156 strain provided by the invention is SEQ ID NO. 3.
The Riboprinter fingerprint of the strain of Lactobacillus rhamnosus VHProbi V6156 provided by the invention is shown in figure 4; the RAPD fingerprint is shown in FIG. 5, and the rep-PCR fingerprint is shown in FIG. 6.
The invention also provides a probiotic preparation, which comprises living bacteria of Lactobacillus rhamnosus VHProbi V6156 strain and/or fermentation products thereof.
The probiotic formulation further comprises a combination of any one or more of lactobacillus acidophilus, bacillus coagulans, pediococcus pentosaceus, lactobacillus rhamnosus, lactobacillus paracasei, bifidobacterium lactis, lactobacillus salivarius, lactococcus lactis, lactobacillus reuteri, lactobacillus casei, lactobacillus helveticus, enterococcus, lactobacillus bulgaricus, lactobacillus grignard, lactobacillus fermentum, streptococcus thermophilus, lactobacillus crispatus.
The live bacterial amount of the lactobacillus rhamnosus VHProbi V6156 strain in the probiotic preparation is not less than 10 8 CFU/g.
The lactobacillus rhamnosus VHProbiV6156 strain provided by the invention has a very strong degradation effect on intestinal indole, can significantly degrade indole in vitro, and has a degradation rate of 52.34 +/-4.82% in 48 hours. The strain also has strong antioxidant capacity and cholesterol degradation capacity, DPPH clearance rate reaches 11.38%, lipid peroxidation inhibition rate is 11.75%, and cholesterol degradation rate reaches 20.39%.
The lactobacillus rhamnosus VHProbi V6156 strain has stronger tolerance to artificial intestinal gastric juice and contains bile salt hydrolase; is sensitive to common antibiotics such as erythromycin and ampicillin, does not generate hemolysin, can not dissolve blood cells, and has good biological safety.
The lactobacillus rhamnosus VHProbi V6156 strain can be used alone or in combination with various probiotics, is used for maintaining human health, and has wide application prospect.
Drawings
FIG. 1 is a graph of the growth of Lactobacillus rhamnosus VHProbi V6156 in an indole-containing medium;
FIG. 2 is a photograph of a colony morphology of Lactobacillus rhamnosus VHProbi V6156;
FIG. 3 is a carbon source metabolism map of Lactobacillus rhamnosus VHProbi V6156;
FIG. 4 is a fingerprint of Lactobacillus rhamnosus VHProbi V6156 Riboprinter;
FIG. 5 is a RAPD fingerprint of Lactobacillus rhamnosus VHProbi V6156;
FIG. 6 is a rep-PCR fingerprint of Lactobacillus rhamnosus VHProbi V6156;
FIG. 7 is a graph of the bile-salt enzyme activity of Lactobacillus rhamnosus VHProbi V6156.
Detailed Description
The lactobacillus rhamnosus VHProbi V6156 provided by the invention meets the requirement of regulations, can be used as a food raw material source, and has no side effect and excessive risk after long-term administration. The multi-phase taxonomy identification shows that the lactobacillus rhamnosus VPHrobi V6156,6156 is a novel lactobacillus rhamnosus strain. The lactobacillus rhamnosus VHProbi V6156 provided by the invention has the effects of degrading intestinal indole, reducing the content of urotoxin indoxyl sulfate and relieving chronic kidney disease, and can be used singly to degrade indole without being compounded with prebiotics and/or other probiotics, so that the lactobacillus rhamnosus VHProbi V6156 has important application value.
The applicant preserved the screened lactobacillus rhamnosus VHProbi V6156 (Lacticaseibacillus rhamnosus VHProbi V6156) at 10 months 26 of 2022 with the China center for type culture collection of university of martial arts, with a preservation number of CCTCC NO: m20221668.
The screening method of the present invention is not limited to the examples, but known screening methods can be used to achieve the screening purpose, and the screening description of the examples is only illustrative of the present invention and is not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The present invention will be described in detail with reference to specific examples.
Example 1 isolation screening of strains
1. Primary screen
Preparing MRS (Man Rogosa Sharpe) broth: 1L of pure water, 10g of peptone, 10g of beef extract, 5.0g of yeast extract, 5g of sodium acetate, 5g of glucose, 2g of monopotassium phosphate, 1.0mL of tween 80, 2.0g of diamine citrate, 20g of calcium carbonate, 0.58g of magnesium sulfate heptahydrate, 0.25 g of manganese sulfate heptahydrate and pH adjustment of 6.2-6.5.
Preparing MRS agar culture medium: 1LMRS broth 15g agar was added.
Taking 1g of yoghourt, diluting the yoghourt by using sterile normal saline, putting the yoghourt into a sterile sample bag, and beating and uniformly mixing the yoghourt by using a homogenizer; and (3) taking 100 mu L of mixed solution for gradient dilution, coating the mixed solution on an MRS agar medium, and performing anaerobic culture at 37 ℃ for 48 hours, and performing microscopic examination on a single colony after the plate grows. According to the microscopic examination result, the applicant screens out 24 potential lactic acid bacteria, which are respectively named as V1, V2, … … and V24. Picking single colony, performing amplification culture in liquid MRS culture medium, further performing coating separation, purifying for 3-5 times, and separating to obtain pure plates with different colony morphologies; selecting single colony of the pure plate in MRS liquid culture medium, culturing at 37 deg.C overnight, and separating bacterial suspension to obtain primary screening bacterial suspension.
2. Bacterial strain screening of in vitro degradable indole
2.1 Preparation work
Preparing an indole solution: preparing an indole mother solution with the concentration of 30mg/ml by using 70% ethanol, and sterilizing a filter membrane with the concentration of 0.22 mu m for later use. The strain culture medium is diluted to the required concentration when in use.
Strain preparation: lactic acid bacteria streak activation. And taking the frozen and preserved lactobacillus glycerin tube, performing aseptic operation, streaking to an MRS plate, and culturing at 37 ℃ for 48-72 h.
Preparation of a lactic acid bacteria cell solution: after single colony is grown on the plate, the plate is aseptically picked up to MRS broth, and the plate is subjected to stationary culture at 37 ℃ for 24 hours. The fresh MRS broth was inoculated at 1% inoculum size, about 6ml per strain.
2.2 Resistance test of strains to different concentrations of indole.
Indole is diluted to 0, 50, 100, 150, 200, 300, 400 and 600 mu g/ml by a strain culture medium, strains are inoculated according to 1% of inoculation amount respectively, and the growth curve of the strains is monitored by an enzyme-labeling instrument. The results show that the activity of the strain is not affected when the low-concentration indole is contained in the culture medium, even the strain growth is slightly promoted, when the indole content in the culture medium is close to 300 mu g/ml, the activity of the strain is inhibited, and meanwhile, according to the data, 0.25-2.6mM of human intestinal canal or fecal indole is shown to be about 45-468 mu g/ml, so that 300 mu g/ml can be used as the indole addition concentration for verifying the indole degradation capacity of the strain.
2.3 Detection of indole metabolizing ability of the strain:
Adding indole into fresh culture medium containing 1% strain to make its final concentration 300 mug/ml, adding indole for 0h, 24h and 48h, respectively taking 1.5ml culture medium to new EP tube, centrifuging for 3min, collecting supernatant, and detecting indole content in supernatant by using indole detection kit. The operation method and the result judgment are specifically described in Indole Assay Kit specifications.
Indole degradation rate calculation: indole degradation rate = (initial concentration-final concentration)/initial concentration × 100%.
The result shows that the highest indole degradation rate in 24 potential lactic acid bacteria obtained by the separation of the invention is a V6 strain, and the indole degradation rate in 24h is 30.72+/-2.17%; the degradation rate of the indole is up to 51.08 +/-4.03% after 48 hours. The growth curves of the V6 strain in different concentrations of indole are shown in FIG. 1.
Example 2 identification of strains
1. Colony morphology identification
The colony morphology of V6 strain on MRS agar culture medium is shown in figure 2, the colony is cream white, opaque, has a diameter of 0.5-1mm, has smooth surface, is characterized by rod shape, and has single, paired or clustered cells.
2. Identification of physiological and biochemical characteristics
The inoculum preparation in this example was as follows: under the aseptic condition, a proper amount of fresh V6 bacterial liquid is taken, centrifuged at 5000rpm/min for 5min times, washed with PBS buffer for 2 times, and then the bacterial cells are diluted by 50 times after being weighted by the same volume of PBS buffer as an inoculation liquid.
2.1 Salinity tolerance test
Under aseptic conditions, 190. Mu.L of BSM liquid medium with salt concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8% was added to the 96-well plate, respectively, 3 replicates of each salt concentration, and then 10. Mu.L of inoculum was added thereto, and the wells without inoculation were used as controls. 50. Mu.L of autoclaved paraffin oil was added to each well to prevent evaporation of water during the culture. Culturing at 37deg.C, and observing whether the culture medium becomes turbid. The results showed that the V6 strain had a maximum tolerated salt concentration of 7%.
2.3 Carbon Source metabolism test
And C, carrying out carbon source metabolism experiments on the V6 strain by using an API 50CHL kit, wherein the concrete reference of the description of the API 50CHL kit is the experimental method and the result interpretation. The identification result of the V6 strain is as follows: % id=99.9 and T value=0.89, api results are lactobacillus rhamnosus, see figure 3 for results.
2.4 Glucose acid and gas production test
The medium formulation used in this example is as follows:
Peptone 0.5g; 0.3g of yeast extract; tween 80.1 ml; 0.5ml of salt solution A; 0.5ml of salt solution B; 0.5g of sodium acetate; glucose 2.5g; 0.05mL of 2% bromocresol green (w/v); distilled water 100ml; the pH is 6.8-7.0.
The prepared culture medium was dispensed into large tubes containing inverted small tubes, 3 mL/tube, and autoclaved at 121℃for 15min.
Salt solution A: KH 2PO4 10g、K2HPO4 1.0.0 g was dissolved in distilled water to a volume of 100mL.
Salt solution B: mgSO 4·7H2O 11.5g、MnSO4·2H2O 2.4g、FeSO4·7H2 O0.68 g was dissolved in distilled water and the volume was set to 100mL.
Under aseptic condition, inoculating the inoculating solution with 10% inoculating amount, inoculating the culture medium without inoculating bacteria as control, sealing the top with 2mL sterile liquid paraffin, culturing at 37deg.C for 24 hr, and observing whether the color of the culture medium changes.
The results show that: after 24h of culture at 37 ℃, the culture medium turns from green to yellow, and no gas exists in the test tube, which indicates that the V6 strain ferments glucose to produce acid and does not produce gas.
3. Molecular biological identification
3.1 16S rDNA Gene sequence analysis
3.1.1 Genomic DNA extraction
Reference was made to the Tiangen bacterial genomic DNA extraction kit (catalog number: DP 302).
3.1.2, 16S rDNA Gene amplification
Primer sequence:
27F：AGAGTTTGATCCTGGCTCA（SEQ ID NO:1）；
1492R：GGTTACCTTGTTACGACTT（SEQ ID NO:2）。
the 16s rDNA sequence of the V6 strain was obtained by sequencing and the sequences were aligned in NCBI database to preliminarily determine that the V6 strain was Lactobacillus rhamnosus.
The 16s rDNA sequence SEQ ID NO. 3 of the V6 strain was obtained by sequencing and the sequences were aligned in NCBI database to determine that the V6 strain was Lactobacillus rhamnosus.
The specific sequence of 16s rDNA is as follows:
GCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCACGCCGACAACAGTTACTCTGCCGACCATTCTTCTCCAACAACAGAGTTTTACGACCCGAAAGCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATCATTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATACGCCGCGGGTCCATCCAAAAGCGATAGCTTACGCCATCTTTCAGCCAAGAACCATGCGGTTCTTGGATTTATGCGGTATTAGCATCTGTTTCCAAATGTTATCCCCCACTTAAGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCACTCGTTCAAAATTAAATCAAGATGCAAGCACCTTTCAATAATCAGAACTCGTTCGACTGC.
3.2 Riboprinter fingerprint
The purified single colony is dipped from an agar culture medium plate by a fungus taking rod, the single colony is placed into a sample tube with buffer solution, the single colony is stirred by a hand-held stirrer to be suspended in the buffer solution, then a sample frame is placed into a heater for inactivation and then placed into a Riboprinter system, and a bacterial identification result is obtained after DNA preparation, film transfer, imaging detection and data processing are carried out on the sample. The identification result shows that the V6 strain is Lactobacillus rhamnosus, and the Riboprinter fingerprint result is shown in FIG. 4.
3.3 RAPD and rep-PCR fingerprint identification
3.3.1 RAPD finger print identification
Primer sequence: GAGGGTGGCGGTTCT (SEQ ID NO: 4).
Table 1: RAPD reaction System Table
Reaction components Volume of
TaqDNA polymerase (5U/. Mu.L) 0.2 μl
10 XBuffer (containing Mg2+) 2 μl
Primer (10 mu M) 1 μl
dNTPs（2.5 mM） 0.8 μl
DNA template 2 μl
Sterile double distilled water 14 μl
Total volume of 20 μl
1.5% Agarose gel plates were prepared, DL2000DNA MARKER was used as a result control, 100V was regulated for 80min, and finally the electropherograms were detected using a gel imaging system. RAPD finger-prints of V6 strain are shown in FIG. 5.
3.3.2 Rep-PCR finger print
Primer sequence: CTACGGCAAGGCGACGCTGACG (SEQ ID NO: 5).
Reaction system of rep-PCR
Table 2: table of the reaction System of rep-PCR
Reaction components Volume of
R TaqDNA polymerase 0.2 μl
10X Ex Taq DNA Buffer (with Mg 2+) 2 μl
Primer (10 mu M) 1 μl
dNTPs（2.5 mM） 2 μl
DNA template 2 μl
Sterile double distilled water 12.8 μl
DL2000 DNA MARKER was used as a result control, voltage 100V, and electrophoresis time 80min to detect the amplification result. The rep-PCR fingerprint of the V6 strain is shown in FIG. 6.
3.4 Whole genome sequencing
Fresh bacterial liquid was inoculated into MRS liquid medium at an inoculum size of 0.1%, cultured at 37℃for 20 hours, centrifuged at 8000rpm for 10min, and the bacterial cells were collected. The bacterial cells are sent to a sequencing center to obtain the whole genome sequence of the bacterial cells, and the genome sequence is uploaded to NCBI gene database, and the GenBank accession number is CP134215.
In summary, by combining the colony morphology and the physiological and biochemical characteristic results of the V6 strain and the molecular biological identification results, the V6 strain is determined to be a novel strain of Lactobacillus rhamnosus, and the novel strain is named Lactobacillus rhamnosus VHProbi V6156 (Lacticaseibacillus rhamnosus VHProbi V6156).
Example 3 test of resistance of Lactobacillus rhamnosus VHProbi V6156 to artificial gastric juice and artificial intestinal juice
1. Preparation of artificial gastric juice
5G of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl are weighed respectively, 1000mL of distilled water is added, pH is adjusted to 3.0 by dilute hydrochloric acid, and then sterilization is carried out for 20min at 115 ℃. Then 3.2g of pig mucosa pepsin is added before use, the pig mucosa pepsin is uniformly shaken and dissolved, and the mixture is placed in a water bath shaker at 37 ℃ for warm water bath for 1 hour so as to simulate the temperature of a human body.
2. Preparation of artificial intestinal juice
5G of peptone, 2.5g of yeast extract, 1g of glucose, 6.8g of KH 2PO4 and 3.0g of ox gall salt are respectively weighed, 77mL of 0.2mol/L NaOH solution is added, the volume is fixed to 1000mL, the pH is regulated to 6.8+/-0.1 by dilute hydrochloric acid or sodium hydroxide solution, and the mixture is sterilized for 20min at 115 ℃. Then adding 1g of pancreatin before use, shaking to dissolve, and placing in a water bath shaker at 37 ℃ for warm water bath for 1h to simulate the temperature of human body.
3. Experimental method
2ML of fresh bacterial liquid is taken, the bacterial liquid is collected by centrifugation at 5000rpm/min for 5min, the bacterial liquid is washed 3 times by physiological saline, and then 2mL of physiological saline is used as inoculation liquid for resuspension. 1mL of the inoculation liquid is taken and added into 24mL of artificial gastric juice or intestinal juice, and the mixture is placed on a water bath shaking table (200 rpm/min) at 37 ℃ for 3 hours, 1mL of sample is taken, and the viable bacteria amount is detected.
The viable bacteria counting method is used for measuring the bacterial amount according to national standard GB 4789.35-2016-lactobacillus test for food microorganism test, and the viable bacteria amount (Log CFU/mL) of the strain after being digested by artificial gastric juice or intestinal juice is shown in Table 3.
Table 3: viable bacteria scale after digestion of artificial gastrointestinal fluids
Before digestion After artificial gastric juice digestion After digestion of the artificial intestinal juice
7.84 ±0.07 7.94 ±0.09 7.39±0.05
As shown in Table 3, the live bacterial amount of the lactobacillus rhamnosus VHProbi V6156 screened by the method is basically unchanged after the lactobacillus rhamnosus VHProbi V is digested by artificial gastric juice, and after the lactobacillus rhamnosus is digested by artificial intestinal juice for 3 hours, the bacterial amount is reduced by only 0.45 Log, which indicates that the strain has strong tolerance to the artificial gastric juice.
Example 4 antibiotic resistance test of Lactobacillus rhamnosus VHProbi V6156
The specific results of the minimal inhibitory concentration MIC values of antibiotics for Lactobacillus rhamnosus VHProbi V6156,6156 are shown in Table 4.
Table 4: antibiotic MIC value data sheet for lactobacillus rhamnosus VHProbi V6156
MIC units μg/mL.
From the results shown in Table 4, the Lactobacillus rhamnosus VHProbi V6156,6156 provided by the invention is sensitive to common antibiotics such as erythromycin and ampicillin, and has good biological safety.
Example 5 intestinal cell adhesion test of Lactobacillus rhamnosus VHProbi V6156
Caco-2 cells were inoculated in six-well plates at an inoculum size of 2X 10 6 cells/well, and cultured in a carbon dioxide incubator for 24 hours for cell adhesion experiments; strains at stationary phase were resuspended to 1X 10 8 CFU/mL with MRS medium; 1mL of the strain is taken and added into a six-hole plate attached to the existing cells, and the culture is carried out for 2 hours in a carbon dioxide incubator; repeatedly washing with PBS for 3 times to remove non-adhering bacteria; adding 500 mu l of pancreatin for digestion for 3 minutes, adding 1.5mL of cell culture liquid for stopping digestion, repeatedly blowing, collecting the obtained solution into a sterile EP tube, and carrying out gradient dilution of 10 times, 100 times, 1000 times and 10000 times on the collected solution, and plating and counting. Cells from the blank group were counted simultaneously. The adhesion ability of the test strain was calculated according to the following formula:
adhesion capacity (CFU/cells) =total number of bacteria adhered per culture well/total number of cells per culture well.
The results show that: the adhesion capacity of lactobacillus rhamnosus VHProbi V6156 is 1.93 and the standard deviation is 0.35%.
Example 6 antioxidant function assay of Lactobacillus rhamnosus VHProbi V6156
1. Determination of DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) removal ability of Strain
1ML of a bacterial suspension of Lactobacillus rhamnosus VHProbi V6156,6156 is taken, 1mL of a 0.4 mM solution of DPPH free radical is added, the mixture is uniformly mixed and then placed at room temperature for shading reaction for 30min, then an absorbance A sample of the sample at the wavelength of 517nm is measured, and the sample is measured for 3 times of parallelism. The control samples were zeroed with equal volumes of PBS and DPPH ethanol mixed solution, and with equal volumes of Lactobacillus rhamnosus VHProbi V6156 bacterial suspension and ethanol mixed solution.
The clearance is calculated according to the following formula:
Clearance% = [1- (a Sample of -A Blank space ）/A Control ] ×100%).
The results are shown in Table 5.
Table 5: DPPH free radical clearance data sheet
Strain Clearance rate of Standard deviation of
Lactobacillus rhamnosus VHProbi V6156 11.38% 2.65%
2. Identification of strains by anti-lipid peroxidation experiments
Culturing Lactobacillus rhamnosus VHProbi V6156 in MRS liquid culture medium at 37deg.C for 24 hr, transferring for 3 generations, centrifuging at 6000 r/min at 4deg.C for 10min, and collecting supernatant to obtain fermentation supernatant. The collected cells were centrifuged at 6000 r/min for 10min with PBS buffer (pH 7.4), and washed 3 times. The cells were resuspended in PBS buffer and the cell concentration was adjusted to 1.0X10 9 cells/mL to obtain a bacterial suspension. The bacterial suspension is subjected to ultrasonic crushing for 20 minutes by an ultrasonic crusher to obtain intracellular extracts.
Preparation of linoleic acid emulsion: 0.1mL linoleic acid, 0.2mL Tween 20, 19.7mL deionized water.
0.5 Adding 1mL linoleic acid emulsion, 1 mLFeSO 4 (1%), 0.5 mL sample, 37 ℃ water bath 1.5 h, 0.2 mL TCA (4%), 2 mL TBA (0.8%), 100 ℃ water bath 30 min, rapidly cooling, 4000 rpm/min centrifuging 15 min, collecting supernatant, and measuring absorbance at 532 nm to obtain A; the control group was A0 with 0.5 mL distilled water instead of the sample.
Inhibition rate/% = (A0-a)/a0×100%.
Wherein: a is absorbance of a sample group; a0 is absorbance of the control group. The results are shown in Table 6.
Table 6: table of the inhibition rate of lactobacillus rhamnosus VHProbi V6156 against lipid peroxidation
Lactobacillus rhamnosus VHProbi V6156 Inhibition rate Standard deviation of
Fermentation supernatant 11.75% 1.13%
Thallus 2.48% 0.52%
Example 7 Lactobacillus rhamnosus VHProbi V6156 in vitro cholesterol degradation assay
1. Qualitative assay of bile salt enzyme Activity
To freshly prepared MRS broth was added 0.2% TCA, 0.2% sodium thioglycolate, 0.37 g/L CaCl 2, and 1.5% agar. Sterilizing at 121deg.C for 15min, pouring into a flat plate until MRS is solidified and placed into an anaerobic tank for use. The sterile filter paper sheet was placed uniformly in a prepared plate, 10 mu L O of freshly cultured bacterial liquid was added dropwise to the filter paper sheet by a pipette, the plate was placed in an anaerobic jar again, and the result was observed after culturing at 37℃for 72 h.
As a result, as shown in FIG. 7, a calcium ring appeared around the filter paper sheet, indicating that the bile salt enzyme activity of the strain V6156 was positive.
2. Cholesterol degradation test
Preparing a cholesterol micelle solution: 1g of cholesterol is accurately weighed, dissolved in absolute ethyl alcohol, and is fixed to a volume of 100 mL, and is subjected to filtration sterilization by a microporous filter membrane of 0.22 mu m under the aseptic condition.
10.0 G parts of peptone, 10.0 g parts of beef extract, 5.0 parts of yeast extract g parts of diammonium hydrogen citrate 2.0 parts of g parts of glucose 20.0 parts of g parts of Tween 80.0 mL, 5.0 parts of sodium acetate g parts of magnesium sulfate 0.1 parts of g parts of magnesium sulfate 0.05 parts of g parts of manganese sulfate 2.0 parts of dipotassium hydrogen phosphate 2. 2.0 g parts of bile salt 1g of distilled water 1000mL parts of the beef extract, adjusting the pH value to 7.3 and sterilizing the beef extract at 115 ℃ for 30 minutes, and then adding a cholesterol solution to ensure that the final concentration of cholesterol is 0.1 percent.
Inoculating fresh bacterial liquid according to 0.1% of inoculation amount, standing at 37 ℃ for 48 hours, taking 0.2mL of bacterial liquid, adding 1.8mL of absolute ethyl alcohol, uniformly mixing, standing for 10 minutes, centrifuging at 3000 r for 5 minutes, and taking supernatant for measuring cholesterol content. Cholesterol measurement method according to GB/T5009.128-2003 < measurement of cholesterol in food >.
The results show that: the degradation rate of the lactobacillus rhamnosus VHProbiV6156 on cholesterol reaches 20.39% (which is data without bile salt).
In conclusion, the lactobacillus rhamnosus VHProbi V6156 provided by the invention is sensitive to common antibiotics, does not produce hemolysin, and has good biological safety. In vitro experiments prove that the lactobacillus rhamnosus VHProbi V6156 has strong degradation capability on indole, can remove uremic toxins and relieve chronic kidney diseases.
The lactobacillus rhamnosus VHProbi V6156 strain provided by the invention can be used alone as probiotics, and can also be used in combination with probiotics such as lactobacillus acidophilus, bacillus coagulans, pediococcus pentosaceus, lactobacillus paracasei, bifidobacterium lactis, lactobacillus salivarius, lactococcus lactis, lactobacillus reuteri, lactobacillus casei, lactobacillus helveticus, enterococcus, lactobacillus bulgaricus, lactobacillus grignard, lactobacillus fermentum, streptococcus thermophilus, lactobacillus crispatus and the like, so that the lactobacillus rhamnosus can be used for maintaining human health and has a wide application prospect.

Claims (6)

1. The lactobacillus rhamnosus is characterized in that the preservation number of the lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) is CCTCC NO: M20221668.
2. The lactobacillus rhamnosus of claim 1 wherein the 16s rDNA sequence of lactobacillus rhamnosus is SEQ ID No. 3.
3. Use of the lactobacillus rhamnosus of claim 1 for the preparation of an indole-degrading product.
4. The use according to claim 3, wherein the indole is an indole in the intestinal tract.
5. Use of lactobacillus rhamnosus as claimed in claim 1 in the preparation of an antioxidant preparation.
6. Use of the lactobacillus rhamnosus of claim 1 for the preparation of a cholesterol-degrading product.
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