CN116478874B - Lactobacillus paracasei for improving chronic low-grade inflammation and application thereof - Google Patents
Lactobacillus paracasei for improving chronic low-grade inflammation and application thereof Download PDFInfo
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- CN116478874B CN116478874B CN202310350808.1A CN202310350808A CN116478874B CN 116478874 B CN116478874 B CN 116478874B CN 202310350808 A CN202310350808 A CN 202310350808A CN 116478874 B CN116478874 B CN 116478874B
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- grade inflammation
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Abstract
The invention relates to a Lactobacillus paracasei for improving chronic low-grade inflammation and application thereof, wherein the Lactobacillus paracasei for improving chronic low-grade inflammation is named as Lactobacillus paracasei Lactobacillus paracasei LC strain, the preservation number is CGMCC No.24411, and the preservation date is 2022, 02 and 21. The strain can improve chronic low-grade inflammation, ovarian dysfunction and type2diabetes of organism.
Description
Technical Field
The invention belongs to the technical field of microbial culture, relates to lactobacillus paracasei for improving chronic low-grade inflammation and application thereof, and in particular relates to lactobacillus paracasei Lactobacillus paracasei LC strain, a culture containing the same, a probiotic containing the same, and application of the same in preparation of medicines for preventing, improving or treating chronic low-grade inflammation, ovarian dysfunction or type2 diabetes.
Background
Chronic low-grade inflammation (CLGI) is a non-specific, sustainable low-grade inflammatory state, also known as chronic systemic low-grade inflammation (chronic systematic low-grade inflammation, CSLGI) due to its presence in multiple tissues/organs throughout the body. Studies have shown a broad link between chronic low grade inflammation and cardiovascular disease, diabetes, ovarian dysfunction disease, alzheimer's disease, cancer. There is growing evidence that inhibiting chronic low grade inflammation improves ovarian function, while chronic low grade inflammation plays a key role in the development and progression of ovarian dysfunction diseases such as ovarian cancer, polycystic ovary syndrome, and the like.
Diabetes (Diabetes mellitus, DM) is a complex metabolic disorder of the endocrine system, and with increased living standard, altered dietary structure and lifestyle, the proportion of Type2diabetes (Type 2diabetes mellitus, T2 DM) in diabetics is high, and Type2diabetes causes serious complications and higher disability rate to patients. There is also a significant association between systemic inflammatory response and T2DM, a number of inflammatory response factors playing a key role in the development, progression and complications of T2DM, but it is not known that chronic low-grade inflammation can lead to the onset of T2DM, or serve as a pathological hallmark only. Clinically, the multi-purpose medicine for treating the chronic low-grade inflammation and the T2DM is intervened, and the toxic and side effects of the medicine are very easy to influence the life quality of patients. And the long-term conservation treatment can only temporarily relieve symptoms, is difficult to radically treat, can even cause exacerbation of symptoms or other complications, and has unsatisfactory long-term curative effect.
Probiotics are active microorganisms that can be colonized in the human body to change the flora of a certain part of the host, thereby being beneficial to the health of the host. At present, probiotics have important roles in enhancing immune functions, improving gastrointestinal functions, metabolic diseases and the like, and can play a beneficial role in reducing chronic low-grade inflammation and T2DM treatment. Thus, the use of probiotics to alleviate or assist in the treatment of chronic low grade inflammation and T2DM is highly beneficial to the physical and mental health of the patient.
However, the microbial agents used in the prior art for reducing chronic low-grade inflammation are limited, and the effect of these agents on reducing chronic low-grade inflammation still needs to be further improved. Therefore, how to provide a microbial preparation with good effect of improving or treating chronic low-grade inflammation, which can assist in treating ovarian dysfunction disease or type2diabetes and does not cause adverse reaction to patients in the treatment process, has become a problem to be solved by the technicians in the field.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide lactobacillus paracasei for improving chronic low-grade inflammation and application thereof, in particular to lactobacillus paracasei Lactobacillus paracasei LC strain, a culture containing the same, a probiotic containing the same and application of the same in preparing medicines with the effects of preventing, improving or treating chronic low-grade inflammation, ovarian dysfunction or type2 diabetes.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a lactobacillus paracasei for improving chronic low-grade inflammation, which is named as lactobacillus paracasei Lactobacillus paracasei LC strain, and has a preservation number of CGMCC No.24411 and a preservation date of 2022, 02 month and 21 days.
The invention separates and stores a new lactobacillus paracasei capable of improving chronic low-grade inflammation from human breast milk samples, which is named as lactobacillus paracasei Lactobacillus paracasei LC strain, and the strain can improve chronic low-grade inflammation, ovarian dysfunction and type2diabetes of organisms. The concrete steps are as follows: (1) Reducing the levels of proinflammatory cytokines IL-1 beta, IL-6 and TNF-alpha in ovarian tissues of a chronic low-grade inflammation animal model, and improving the level of anti-inflammatory cytokines IL-10 in ovarian tissues; (2) Can improve the E2 and AMH reduction in the rat serum induced by LPS, and inhibit the FSH and LH elevation in the rat serum induced by LPS; (3) Has good adjuvant therapeutic effect on blood sugar concentration of type2diabetes, and can relieve inflammatory factors generated by human body due to hyperglycemia. Therefore, the lactobacillus paracasei Lactobacillus paracasei LC strain can be used for preparing medicines for preventing, improving or treating chronic low-grade inflammation, ovarian dysfunction or type2diabetes mellitus.
In addition, the lactobacillus paracasei is a probiotic, so that the lactobacillus paracasei LC24 obtained by screening is high in safety when being used for preparing medicines with the effects of preventing, improving or treating chronic low-grade inflammation, ovarian dysfunction or type2 diabetes; and it is not prone to developing drug resistance.
The screening steps of the lactobacillus paracasei LC24 related to the invention are as follows:
(1) Selecting a human breast milk sample, performing 10-time gradient dilution with physiological saline with the mass concentration of 0.9%, diluting for 3 times, coating on an MRS solid culture medium (the culture medium contains an antibiotic mupirocin lithium salt which can inhibit most other strains except bifidobacteria, medicines are purchased from Haibo organisms), culturing at 37 ℃ for 48 hours, selecting bacterial colonies with different forms, performing streak purification on the surface of the MRS solid culture medium, selecting single bacterial colonies, performing expanded culture with the MRS liquid culture medium at 37 ℃, and preserving with glycerol with the mass concentration of 30%.
(2) And (3) carrying out in-vitro physiological characteristic test on the preserved single strains, screening out a single strain with the best gastrointestinal fluid tolerance (artificial simulation), HT-29 cell adhesion capability, bacteriostasis capability and functional oligosaccharide utilization capability, and identifying the single strain.
In a second aspect, the present invention provides a culture of lactobacillus paracasei for ameliorating chronic low grade inflammation, characterized in that the culture is prepared by a process comprising: the lactobacillus paracasei Lactobacillus paracasei LC strain of the first aspect is inoculated into a medium and cultured at 30-40 ℃ (e.g., 30 ℃, 32 ℃, 35 ℃, 36 ℃,37 ℃, 38 ℃ etc.) for 12-30 hours (e.g., 15 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 30 hours, etc.).
Other specific point values within the above numerical ranges are all selectable, and will not be described in detail herein.
The formulation composition of the medium is exemplifiedComprising the following steps: peptone, beef extract, glucose, sodium acetate, yeast powder, diammonium hydrogen citrate, K 2 PO 4 ·3H 2 O、MgSO 4 ·7H 2 O、MnSO 4 L-cysteine, tween 80.
In a third aspect, the present invention provides a probiotic having the efficacy of preventing, ameliorating or treating chronic low grade inflammation, the probiotic comprising lactobacillus paracasei Lactobacillus paracasei LC strain according to the first aspect.
The lactobacillus paracasei Lactobacillus paracasei LC strain related to the invention can be applied to related products alone or in combination with other strains.
In a fourth aspect, the invention provides a probiotic having the efficacy of preventing, ameliorating or treating ovarian dysfunction, the probiotic comprising lactobacillus paracasei Lactobacillus paracasei LC strain according to the first aspect.
The lactobacillus paracasei Lactobacillus paracasei LC strain related to the invention can be applied to related products alone or in combination with other strains.
In a fifth aspect, the present invention provides a probiotic having the efficacy of preventing, ameliorating or treating type2diabetes, the probiotic comprising lactobacillus paracasei Lactobacillus paracasei LC strain according to the first aspect.
The lactobacillus paracasei Lactobacillus paracasei LC strain related to the invention can be applied to related products alone or in combination with other strains.
In any of the above probiotics, the viable count of the Lactobacillus paracasei Lactobacillus paracasei LC strain is not less than 1×10 8 CFU/mL or 1X 10 8 CFU/g, e.g. 1X 10 8 CFU/mL、2×10 8 CFU/mL、5×10 8 CFU/mL、8×10 8 CFU/mL、1×10 9 CFU/mL、5×10 9 CFU/mL、1×10 10 CFU/mL, etc., other specific point values within the numerical range are selected, and no longerAnd the details are as follows.
In any of the above probiotics, the formulation of the probiotics comprises freeze-dried powder, capsules, tablets or granules. The probiotic agent can be freeze-dried powder, and the freeze-dried powder can be further prepared into capsules, tablets or granules and other dosage forms.
In any of the above probiotics, the probiotic further comprises a protective agent and/or a co-additive.
Preferably, the protective agent comprises skim milk powder.
The auxiliary additive comprises any one or a combination of at least two of fructo-oligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, isomalto-oligosaccharide, soybean oligosaccharide, inulin, spirulina, arthrospira, coriolus versicolor polysaccharide, stachyose, polydextrose, alpha-lactalbumin or lactoferrin.
In any of the above probiotics, the probiotics further comprises lactobacillus plantarum Lactobacillus plantarum Lp strain; the preservation number of the lactobacillus plantarum Lactobacillus plantarum Lp strain is CGMCC No.10453, and the preservation date is 2015, 01 and 27.
The invention also creatively discovers that the lactobacillus paracasei Lactobacillus paracasei LC strain can be compounded with the lactobacillus plantarum Lactobacillus plantarum Lp strain for use in preventing, improving or treating chronic low-grade inflammation, ovarian dysfunction or type2diabetes, has an effect remarkably superior to that of a single microbial inoculum or other compound formulas, and shows that the LC24 strain and the Lp90 strain have a synergistic effect on the effects.
Further preferably, the ratio of the viable count of the lactobacillus paracasei Lactobacillus paracasei LC strain to the lactobacillus plantarum Lactobacillus plantarum Lp strain is (1-3): (1-3), for example, 1:3, 1:2, 1:1, 2:1, 3:1, etc., and other specific values within the numerical range can be selected, so that no further description will be given here.
In the composite probiotic, the two strains have better synergistic effect when meeting the specific mass proportion relation.
In a sixth aspect, the invention provides the use of a lactobacillus paracasei Lactobacillus paracasei LC strain according to the first aspect or a culture according to the second aspect or a probiotic according to the third aspect in the manufacture of a medicament for the prevention, amelioration or treatment of chronic low-grade inflammation.
In a seventh aspect, the invention provides the use of a lactobacillus paracasei Lactobacillus paracasei LC strain according to the first aspect or a culture according to the second aspect or a probiotic according to the fourth aspect in the manufacture of a medicament for the prevention, amelioration or treatment of ovarian dysfunction.
In an eighth aspect, the invention provides the use of a lactobacillus paracasei Lactobacillus paracasei LC strain according to the first aspect or a culture according to the second aspect or a probiotic according to the fifth aspect in the manufacture of a medicament having the efficacy of preventing, ameliorating or treating type2 diabetes.
Compared with the prior art, the invention has the following beneficial effects:
the invention separates and stores a new lactobacillus paracasei capable of improving chronic low-grade inflammation from human breast milk samples, which is named as lactobacillus paracasei Lactobacillus paracasei LC strain, and the strain can improve chronic low-grade inflammation, ovarian dysfunction and type2diabetes of organisms. The concrete steps are as follows: (1) Reducing the levels of proinflammatory cytokines IL-1 beta, IL-6 and TNF-alpha in ovarian tissues of a chronic low-grade inflammation animal model, and improving the level of anti-inflammatory cytokines IL-10 in ovarian tissues; (2) Can improve the E2 and AMH reduction in the rat serum induced by LPS, and inhibit the FSH and LH elevation in the rat serum induced by LPS; (3) Has good adjuvant therapeutic effect on blood sugar concentration of type2diabetes, and can relieve inflammatory factors generated by human body due to hyperglycemia. Therefore, the lactobacillus paracasei Lactobacillus paracasei LC strain can be used for preparing medicines for preventing, improving or treating chronic low-grade inflammation, ovarian dysfunction or type2diabetes mellitus.
The invention also creatively discovers that the lactobacillus paracasei Lactobacillus paracasei LC strain can be compounded with the lactobacillus plantarum Lactobacillus plantarum Lp strain for use in preventing, improving or treating chronic low-grade inflammation, ovarian dysfunction or type2diabetes, has an effect remarkably superior to that of a single microbial inoculum or other compound formulas, and shows that the LC24 strain and the Lp90 strain have a synergistic effect on the effects.
Detailed Description
In order to further describe the technical means adopted by the present invention and the effects thereof, the following describes the technical scheme of the present invention in combination with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.
The lactobacillus paracasei Lactobacillus paracasei LC strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.24411, the preservation date of 2022 and 21 days, and the preservation address of North Chen West Lu No.1 and 3 in the Chaoyang area of Beijing city.
The lactobacillus plantarum Lactobacillus plantarum Lp strain which is related to the following is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.10453, the preservation date of 2015, 1 month and 27 days, and the preservation address of North Chen West Lu No.1, 3 in the Chaoyang area of Beijing city.
MRS solid medium used as follows: weighing 10g of peptone, 10g of beef extract, 20g of glucose, 2g of sodium acetate, 5g of yeast powder, 2g of diammonium hydrogen citrate and K 2 PO 4 ·3H 2 O 2.6g、MgSO 4 ·7H 2 O 0.1g、MnSO 4 0.05g, 20g of agar and 0.5g of L-cysteine are dissolved by deionized water, 1mL of Tween 80 is added, the volume is fixed to 1L, and after sterilization and cooling, the mixture is poured into a sterilized culture dish for standby.
MRS liquid medium used as follows: weighing 10g of peptone, 10g of beef extract, 20g of glucose, 2g of sodium acetate, 5g of yeast powder, 2g of diammonium hydrogen citrate and K 2 PO 4 ·3H 2 O 2.6g、MgSO 4 ·7H 2 O 0.1g、MnSO 4 0.05g and 0.5g of L-cysteine are dissolved by deionized water, then 1mL of Tween 80 is added, the volume is fixed to 1L, and the sterilization and cooling are carried out for standby.
DMEM medium was purchased from marsupenario life technologies limited; fetal bovine serum, PBS and trypsin were purchased from Thermo corporation.
The preparation method of the bacterial suspension comprises the following steps: inoculating the required strain into MRS liquid culture medium, culturing at 37deg.C for 18h for activation, and continuously activating for 2 times to obtain activating solution; inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 2% (v/v), and culturing at 37 ℃ for 18h to obtain bacterial solution; centrifuging the bacterial liquid at 8000g for 15min, and re-suspending the bacterial body by using PBS.
Example 1
In this example, a strain of Lactobacillus paracasei was screened for improvement of chronic low-grade inflammation as follows:
(1) Selecting a human breast milk sample, performing 10-time gradient dilution with physiological saline with the mass concentration of 0.9%, diluting for 3 times, coating on an MRS solid culture medium (the culture medium contains an antibiotic mupirocin lithium salt which can inhibit most other strains except bifidobacteria, medicines are purchased from Haibo organisms), culturing at 37 ℃ for 48 hours, selecting bacterial colonies with different forms, performing streak purification on the surface of the MRS solid culture medium, selecting single bacterial colonies, performing expanded culture with the MRS liquid culture medium at 37 ℃, and preserving with glycerol with the mass concentration of 30%.
(2) The following in vitro physiological property tests are carried out on the preserved single bacteria derived from human milk, and a single strain with the best gastrointestinal fluid tolerance (artificial simulation), HT-29 cell adhesion capability, bacteriostasis capability and functional oligosaccharide utilization capability is screened out, specifically as follows:
(2.1) gastrointestinal fluid tolerance test:
gastric juice is simulated manually: preparing 0.5% NaCl solution, adding 0.3% pepsin, adjusting pH to 2.5 with 1mol/L HCl, dissolving completely, and filtering with 0.22 μm microporous membrane for sterilization.
Manually simulating intestinal juice: preparing a 0.5% NaCl solution, adding 0.1% trypsin, adjusting the pH value to 8.0 by using 0.1mol/L NaOH, and filtering and sterilizing by using a 0.22 mu m microporous filter membrane after the solution is fully dissolved for later use.
Anaerobic culturing the strain to be selected in artificial gastric juice or intestinal juice for 3 hours, taking the digestive juice of 0 hours and 3 hours for detecting the viable count, and calculating the survival rate. Strain survival rate (%) =b/a×100%, where a represents the number of viable bacteria (CFU/mL) of the bacteria for 0h, and B represents the number of viable bacteria (CFU/mL) of the bacteria for 3 h.
(2.2) HT-29 cell adhesion Capacity test:
the concentration of digested HT-29 cells was adjusted to 1X 10 5 1mL per well was inoculated into 12 well cell culture plates at 5% CO 2 Incubating in a concentration incubator until cells grow to a monolayer, washing twice with sterile PBS, digesting one well with pancreatin, and counting cells with a blood cell counting plate; 1mL of the strain suspension to be selected is added into other holes respectively (the concentration of the strain suspension is adjusted to be 10) 8 CFU/mL), at 5% CO 2 After incubation for 2h at 37 ℃ in an incubator, washing cells with sterile PBS for 5 times, removing non-adhered bacterial suspension, adding 0.2mL of pancreatin EDTA buffer solution into each hole to digest the cells for 5min, adding 0.8mL of PBS after digestion, blowing evenly, and taking bacterial liquid for dilution and viable bacteria counting.
(2.3) antibacterial ability test:
the inhibition ability of pathogenic bacteria is tested by adopting an oxford cup method: the pathogenic bacterial strains of escherichia coli, salmonella and staphylococcus aureus are respectively inoculated into a liquid beef extract peptone culture medium for overnight culture at 37 ℃ and a rotating speed of 250rpm in a constant-temperature shaking table, so as to prepare pathogenic bacterial suspension. Cooling MRS solid culture medium to about 55deg.C, mixing with pathogenic bacteria suspension at a certain ratio to make the number of viable bacteria of the system be 10 6 The order of CFU/mL is then poured into a flat plate in which an oxford cup is placed in advance rapidly, after the culture medium is cooled and solidified, the oxford cup is taken out, 200 mu L of bacterial liquid of a strain to be tested is poured into each hole, the flat plate is covered lightly and then is placed in a constant temperature incubator at 37 ℃, after the culture is carried out for a proper time, observation is carried out, and the diameter of a bacteriostasis ring is measured by a vernier caliper. The larger the inhibition zone is, the stronger the inhibition capability is.
(2.4) functional oligosaccharide availability test:
sugar-free medium: 1% of peptone, 1% of beef extract, 0.5% of yeast extract, 0.2% of diammonium hydrogen citrate and K 2 HPO 4 0.2％、MgSO 4 0.058％、MnSO 4 0.019%, tween-80 0.1%, L-cysteine hydrochloride 0.1%. Inulin, fructo-oligosaccharide and galacto-oligosaccharide are purchased from Quantum Gao Ke (China) biological Co., ltd. And xylo-oligosaccharide is purchased from Shandong Longli Biotech Co., ltd.
Adding xylooligosaccharide, inulin, fructooligosaccharide and galactooligosaccharide into the sugarless culture medium according to 2% to obtain the culture medium containing oligosaccharide as carbon source. Inoculating the strain to be selected into a culture medium containing oligosaccharide, standing and culturing at a constant temperature of 37 ℃ for 12 hours, and measuring the OD600 absorbance to judge the utilization capacity of the strain to the functional strain.
Example 2
In this example, the strains obtained by screening in example 1 were subjected to morphological identification and 16S rRNA molecular biology identification, as follows:
(1) Morphological identification:
the strain was inoculated in MRS solid medium, cultured at 37℃for 48 hours, and then observed under a microscope. As can be seen from observation, the colony is milky white, round, with a diameter of more than 1.0mm, smooth and raised surface and neat edge.
(2) 16S rRNA molecular biology identification:
taking out the strain preserved at-80deg.C, inoculating into a centrifuge tube containing 20mL MRS liquid culture medium at a ratio of 2%, culturing at 37deg.C for 18 hr, centrifuging at 8000rpm for 10min, removing supernatant, and collecting thallus. Extracting genome of the strain, adding bacterial universal primer for PCR amplification, and delivering amplified product to Shanghai biological engineering Co., ltd for sequencing identification. The strain is subjected to sequencing analysis, and the 16S rDNA sequence of the strain is shown as SEQ ID No.1.
SEQ ID No:1：
ACAATCTTTGTTCACCTTAGACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCTCTTCTCACTCAAGTTTCCCAGTTTCCGATGCGCTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGGGCTGCTGGCACGTAGTTAGCCGTGGCTTTTGTTGGATACCGTCACGCCGACAACAGTTACTCTGCCGACCATTCTTCTCCAACAACAGAGTTTTACGACCCGAAAGCCTTCTTACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATCATCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATACGCCGCGGGTCCATCCAAAAGCGATAGCTTACGCCATCTTTCAGCCAAGAACCATGCGGTCTTGGATCTATGCGGTATTAGCATCTGTTTCCAAATGTTATCCCCCACTTAAGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCACTCGTTCCATGTTGAATCTCGGTGCAAGCACCGATCATCAACGAGAACTCGTTCGACTTGCATGTATAGCACCCGCACTCCA。
The sequences obtained by sequencing were subjected to nucleic acid sequence alignment in GeneBank, and the results showed that the strain was indeed Lactobacillus paracasei.
Example 3
The culture conditions of the Lactobacillus paracasei LC24 were optimized in this example as follows:
inoculating Lactobacillus paracasei LC24 into MRS liquid culture medium, respectively culturing at different temperatures of 10-50deg.C for 48 hr, and measuring OD600 value of the culture solution by enzyme-labeling instrument at intervals during culturing. The results show that the Lactobacillus paracasei LC24 grows optimally at 30-37 ℃, and the stable growth period can be achieved after 18-24 hours of culture.
Example 4
This example demonstrates the gastric acid resistance of Lactobacillus paracasei LC24 as follows:
(1) Preparing artificial gastric juice:
the artificial gastric juice contains 0.20% of NaCl and 0.30% of pepsin by mass fraction, the pH is respectively regulated to 2.0, 2.5 and 3.0 by using HCl, and the artificial gastric juice is filtered and sterilized for standby.
(2) Gastric acid resistance test:
1.0mL of a Lactobacillus paracasei LC24 strain suspension (concentration: 1X 10) 9 CFU/mL, the concentration of bacterial liquid is measured by the method in national standard food safety national Standard microbiological detection of lactic acid bacteria of GB4789.35-2016, respectively, and is mixed with 9.0mL of artificial gastric juice with pH of 2.0, 2.5 and 3.0, and then anaerobic stationary culture is carried out at 37 ℃, sampling is carried out after the beginning (0 h) and the treatment for 3h respectively, the viable count is measured by a pouring culture method, and the survival rate is calculated according to the following formula:
survival (%) =n1/n0×100%,
wherein, N1: viable count after 3 hours of artificial gastric juice treatment; n0: viable count of 0 h. The test results are shown in Table 1.
TABLE 1
| pH of artificial gastric juice | Viable count N0 (0 h) | Viable count N1 (3 h) | Survival (%) |
| 2.0 | 6.82×10 8 | 5.85×10 8 | 85.8. |
| 2.5 | 6.81×10 8 | 6.18×10 8 | 91.4 |
| 3.0 | 6.80×10 8 | 6.33×10 8 | 93.1 |
As can be seen from Table 1, the Lactobacillus paracasei LC24 has good gastric acid resistance, and can be incubated for 3 hours in artificial gastric juice with pH of 2.0, and the survival rate can reach more than 85.8%; incubating in artificial gastric juice with pH of 2.5 for 3 hours, wherein the survival rate can reach more than 91.4%; the survival rate can reach more than 93.1% after the culture in the artificial gastric juice with the pH value of 3.0 for 3 hours. The good acid resistance creates conditions for the preparation of products for improving chronic low-grade inflammation, ovarian function and type2 diabetes.
Example 5
This example demonstrates the cholate resistance of Lactobacillus paracasei LC24, as follows:
the concentration of 1mL was adjusted to 1X 10 9 CFU/mL of Lactobacillus paracasei LC24 was inoculated into 100mL of MRS-THIO medium containing 0.3% bile salts (MRS contains 0.2% sodium thioglycolate), incubated at 37℃for 4h and 6h, and absorbance at 600nm was measured.
The experiment uses cheese bacillus paracasei LC86 with a preservation number of CGMCC No.1.12731 as a control bacterium, and the culture method is the same as that of LC 24. The greater OD600 value indicates a greater resistance to bile salts in Lactobacillus paracasei. The test results are shown in Table 2.
TABLE 2
| Name of the name | OD600(4h) | OD600(6h) |
| Cheese bacillus B111H | 0.23 | 0.19 |
| Lactobacillus paracasei LC24 | 0.37 | 0.32 |
As can be seen from Table 2, after 4H and 6H of incubation, the OD600 of the Lactobacillus paracasei LC24 was 0.37 and 0.32, respectively, which were higher than that of Lactobacillus paracasei B111H, indicating that the Lactobacillus paracasei LC24 was more resistant to bile salts than that of Lactobacillus paracasei B111H.
Example 6
This example demonstrates the ameliorating effect of lactobacillus paracasei LC24 on chronic low grade inflammation and ovarian dysfunction:
180-250g healthy female SD rats were purchased from Shanghai Laek laboratory animal center and all rats were kept for one week under barrier maintenance conditions with controlled light and temperature. (light/dark cycle, 12h/12h; temperature, 22 ℃ + -1 ℃ C.; relative humidity, 55% + -5%). Animal experiments strictly follow the national institutes of health, guidelines for laboratory animal care and use.
(1) Establishing a chronic low-grade inflammation rat model:
according to the common method in the prior art, the chronic low-grade inflammation of rats is induced by intraperitoneal injection of Lipopolysaccharide (LPS) as an animal model, and the chronic low-grade inflammation is induced by intraperitoneal injection of lipopolysaccharide 1 time a day for 14 consecutive days. (ref: intermittent injections of small doses of lipopolysaccharide establish a model of chronic inflammation in rats DOI:10.13375/j. Cnki. Wcjs. 2012.03.027).
(2) Rat grouping and treatment mode:
60 rats were randomly divided into 6 groups (10 per group): normal control group (CTL), untreated chronic low-grade inflammation model group (LPS), chronic low-grade inflammation group treated with lactobacillus paracasei LC24 (LC 24), chronic low-grade inflammation group treated with lactobacillus plantarum Lp90 (Lp 90), chronic low-grade inflammation group treated with lactobacillus paracasei LC24 and lactobacillus plantarum Lp90 (lc24+l90, viable count ratio 1:1), chronic low-grade inflammation group treated with lactobacillus paracasei ATCC25302 and lactobacillus plantarum Lp90 (ATCC 25302+l90, viable count ratio 1:1).
All five groups except the normal control group (CTL) were chronically low-inflammatory rats and the probiotic intervention group was gavaged 1 time (1×10) daily on days 15 to 28 9 CFU/day), whereas normal control group (CTL) and untreated chronic low-grade inflammation model group (LPS) were intragastric at 1mL/100g with physiological saline. All rats were given their respective feed by gavage for 4 weeks. All rats were free to eat and drink throughout the study period.
(3) Sample collection: after the experiment is finished, all rats are fasted for 12 hours and anesthetized by 10% chloral hydrate, the heart is punctured and freshly sampled, and after centrifugation for 10min at 3000r/min, serum is preserved at-80 ℃. Female rats had a estrus cycle of 4-5d, which was divided into 4 phases according to physiological changes: in the pre-estrus, post-estrus and estrus interval, in order to determine and distinguish each estrus stage of a mouse, a vaginal tissue smear method is adopted to observe the diethylstilbestrol treated mouse by respectively carrying out vaginal tissue smear on each stage of an estrus period, so as to explore the development change and morphological characteristics of the cell tissue in the vagina of the mouse in the estrus period and provide a reference for distinguishing the development period stage of the mouse. Vaginal cytology was performed on rats prior to blood withdrawal to determine the estrus cycle each rat was in (the test index was all tested in the last estrus cycle). After blood collection, the rats were sacrificed by quantitative anesthesia, the ovaries were immediately removed, placed in liquid nitrogen for rapid freezing, and transferred to-80 ℃ for preservation.
(4) Determination of ovarian tissue inflammatory factors: the average levels of the pro-inflammatory factors interleukin IL-1 beta (pg/ml), IL-6 (pg/ml) and tumor necrosis factor alpha (TNF-alpha, pg/ml) in ovarian tissue of different estrus cycles were measured in each group of rats using a commercial (Wohan purity Biotechnology Co., wohan, china) ELISA kit. The results are shown in Table 3.
TABLE 3 Table 3
From the data in Table 3, it can be seen that: the cheese bacillus LC24 strain has good effect of inhibiting chronic low-grade inflammation rat ovarian tissue inflammatory factors, namely has excellent effect of improving chronic low-grade inflammation, and can further improve the effect by combining the LC24 strain with the Lp90 strain.
(5) Rat ovarian function evaluation: the levels of estradiol (E2, pg/ml), follicle stimulating hormone (FSH, ng/ml), luteinizing hormone (LH, ng/ml) and anti-muller tube hormone (AMH, ng/ml) in serum from various estrus cycles were measured for each group of rats using a commercial (martial purity biotechnology limited, martial arts, china) ELISA kit to assess ovarian function. The results are shown in Table 4.
TABLE 4 Table 4
From the data in table 4, it can be seen that: the cheese bacillus LC24 strain has good effect of improving the ovarian function of the chronic low-grade inflammation rat, and the effect can be further improved by combining the LC24 strain with the Lp90 strain.
Example 7
This example demonstrates the ameliorating effect of lactobacillus paracasei LC24 on type2diabetes hyperglycemia:
SPF male Kunming mice at 6 weeks of age were purchased from Shanghai Laek laboratory animal center and all mice were kept for one week under barrier maintenance conditions with controlled light and temperature. (light/dark cycle, 12h/12h; temperature, 22 ℃ + -1 ℃ C.; relative humidity, 55% + -5%). Animal experiments strictly follow the national institutes of health, guidelines for laboratory animal care and use.
(1) Model establishment of type2 diabetic hyperglycemia (T2 DM) rat:
healthy rats were numbered and fed 1w adaptively prior to the trial. Before the adaptive feeding is finished, the rats are detected, FPG and fasting serum insulin (FINS) are used for calculating Insulin Sensitivity Index (ISI) and performing an intraperitoneal injection glucose tolerance test, after the adaptive feeding is finished, high-sugar high-fat feed is used for continuously feeding 4w, free water is used for feeding during the period, before the high-sugar high-fat feed feeding is finished, the rats are detected, FINS and ISI are calculated, and an IPGTT test is performed. After the high-sugar high-fat feed is fed, 30mg/kg of 2% STZ is injected into the abdominal cavity according to the weight of a rat in a fasting state, random blood sugar is monitored within 72 hours, 1-2U/kg of insulin is injected into the abdominal cavity when the random blood sugar value is more than or equal to 30.0mmol/L, and glucose solution is injected when the blood sugar value is too low. And detecting FPG after 1w, wherein the fasting blood glucose is more than or equal to 7.0mmol/L, namely the successful T2DM model, and reserving the successful model. The success model detects FPG, pins and calculates ISI.
(2) Rat grouping and treatment mode:
60 rats were randomly divided into 6 groups (10 per group): normal Control (CTL), T2DM without intervention (T2 DM), T2DM treated with lactobacillus paracasei LC24 (LC 24), T2DM treated with lactobacillus plantarum Lp90 (Lp 90), T2DM treated with lactobacillus paracasei LC24 and lactobacillus plantarum Lp90 (LC 24+ Lp90, viable count ratio 1:1), T2DM treated with lactobacillus paracasei ATCC25302 and lactobacillus plantarum Lp90 (ATCC 25302+ Lp90, viable count ratio 1:1).
Five groups, except the normal control group (CTL), all used T2DM model rats, and the probiotic intervention group were gavaged 1 time daily (1 x 10) 9 CFU/day), 0.2mL was orally administered, while the normal control group (CTL) and the T2DM group without intervention were intragastrically irrigated with 0.2mL of physiological saline. All rats were free to eat and drink throughout the study period.
(3) Sample collection: the FINS (fasting insulin levels) was measured weekly. After 6 weeks of intervention, blood was collected by retroorbital bleeding after 12h of fasting, and serum was centrifuged (1150 Xg, 10min,4 ℃). The pancreas, liver and colon contents were stored to-80 ℃ for further analysis.
(4) Oral glucose tolerance test: after 6 weeks of intervention, an Oral Glucose Tolerance Test (OGTT) was performed after 12h of fasting as described previously. The results are shown in Table 5.
(5) Inflammatory factor determination: the method for detecting the levels of TNF-alpha and IL-1β is the same as in example 6. The results are shown in Table 5.
TABLE 5
| Group of | OGTT(mg/dl) | FINS(mIU/L) | IL-1β(pg/ml) | TNF-α(pg/ml) |
| CTL | 7.15 | 5.45 | 18.25 | 123.23 |
| T2DM | 17.65 | 9.59 | 34.12 | 188.25 |
| LC24 | 8.06 | 6.18 | 19.91 | 127.41 |
| Lp90 | 9.43 | 8.57 | 24.89 | 156.35 |
| LC24+Lp90 | 7.56 | 5.53 | 19.73 | 124.59 |
| ATCC25302+Lp90 | 9.23 | 9.45 | 24.65 | 153.24 |
From the data in table 5, it can be seen that: the cheese bacillus LC24 strain related to the invention has good effect of improving the hyperglycemia of type2diabetes, and the LC24 strain and the Lp90 strain can be combined to further improve the effect.
The applicant states that the present invention is illustrated by the above examples as a lactobacillus paracasei for improving chronic low grade inflammation and its use, but the present invention is not limited to the above examples, i.e. it is not meant that the present invention must be practiced in dependence upon the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (10)
1. Lactobacillus paracasei capable of improving chronic low-grade inflammationLactobacillus paracasei) Characterized in that the Lactobacillus paracasei for improving chronic low-grade inflammationLactobacillus paracasei) Named as Lactobacillus paracaseiLactobacillus paracasei) LC24 strain with preservation number of CGMCC No.24411 and preservation date of 2022, 02 and 21.
2. A culture of lactobacillus paracasei that ameliorates chronic low-grade inflammation, the method of preparing the culture comprising: lactobacillus paracasei according to claim 1Lactobacillus paracasei) The LC24 strain is inoculated in a culture medium and cultured at 30-40 ℃ for 12-30 h.
3. A probiotic, comprising Lactobacillus paracasei according to claim 1Lactobacillus paracasei) LC24 strain.
4. A probiotic according to claim 3, characterized in that in the probiotic the lactobacillus paracasei is @ dLactobacillus paracasei) The viable count of LC24 strain is not less than 1×10 8 CFU/mL or 1X 10 8 CFU/g。
5. A probiotic according to claim 3, wherein the probiotic is in a dosage form selected from a lyophilized powder, capsule, tablet or granule.
6. A probiotic according to claim 3, characterized in that the probiotic further comprises a protective agent and/or a co-additive.
7. The probiotic according to claim 6, characterized in that the protective agent comprises skim milk powder;
the auxiliary additive comprises any one or a combination of at least two of fructo-oligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, isomalto-oligosaccharide, soybean oligosaccharide, inulin, spirulina, arthrospira, coriolus versicolor polysaccharide, stachyose, polydextrose, alpha-lactalbumin or lactoferrin.
8. A probiotic according to claim 3, characterized in that the probiotic further comprises lactobacillus plantarum @Lactobacillus plantarum) Lp90 strain; the lactobacillus plantarum is [ ]Lactobacillus plantarum) The preservation number of the Lp90 strain is CGMCC No.10453, and the preservation date is 2015, 01 and 27.
9. The probiotic according to claim 8, characterized in that said lactobacillus paracasei is @ o @ mLactobacillus paracasei) LC24 strain and lactobacillus plantarumLactobacillus plantarum) The ratio of the viable count of the Lp90 strain is (1-3): 1-3.
10. Lactobacillus paracasei according to claim 1Lactobacillus paracasei) Use of LC24 strain or the culture of claim 2 or the probiotic of any one of claims 3-9 in the manufacture of a medicament having the efficacy of preventing, ameliorating or treating ovarian dysfunction or type2 diabetes.
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| KR101869740B1 (en) * | 2017-06-23 | 2018-06-21 | 한국식품연구원 | Novel lactobacillus paracasei strain having advanced glycation end products inhibitory activity effects and use of the same |
| CN110604781A (en) * | 2019-09-23 | 2019-12-24 | 石家庄君乐宝乳业有限公司 | Lactobacillus paracasei N1115-containing bacterial powder capable of preventing and relieving diabetes, and preparation method and application thereof |
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