CN116445346A - Lactobacillus reuteri for improving polycystic ovary syndrome and application thereof - Google Patents
Lactobacillus reuteri for improving polycystic ovary syndrome and application thereof Download PDFInfo
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- CN116445346A CN116445346A CN202310399703.5A CN202310399703A CN116445346A CN 116445346 A CN116445346 A CN 116445346A CN 202310399703 A CN202310399703 A CN 202310399703A CN 116445346 A CN116445346 A CN 116445346A
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- lactobacillus reuteri
- polycystic ovary
- ovary syndrome
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- probiotic
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Abstract
The invention relates to lactobacillus reuteri for improving polycystic ovary syndrome and application thereof, wherein the lactobacillus reuteri for improving polycystic ovary syndrome is named as lactobacillus reuteri Lactobacillus reuteri LR strain, the preservation number is CGMCC No.24408, and the preservation date is 2022, 02 and 21 days. The strain can effectively improve polycystic ovary syndrome and symptoms of related metabolic level disturbance and sex hormone level abnormality, and can be used for preparing products for preventing, relieving or treating polycystic ovary syndrome.
Description
Technical Field
The invention belongs to the technical field of microbial culture, relates to lactobacillus reuteri for improving polycystic ovary syndrome and application thereof, and in particular relates to lactobacillus reuteri Lactobacillus reuteri LR strain, a culture containing the lactobacillus reuteri Lactobacillus reuteri LR strain, a probiotic containing the lactobacillus reuteri, and application of the lactobacillus reuteri in preparation of medicines or health care products with the function of preventing, relieving or treating polycystic ovary syndrome.
Background
Polycystic ovary syndrome (polycystic ovary syndrome, PCOS) is the most common metabolic disease of reproductive endocrine in women of childbearing age. Common clinical manifestations of PCOS include irregular menstruation, high androgen associated manifestations, infertility due to ovulation failure, etc., and may be accompanied by metabolic abnormality symptoms such as obesity, insulin resistance, hyperinsulinemia, glycolipid metabolic disorder, intestinal dysbacteriosis, etc. The long-term disease also can increase the incidence risk of diseases such as type II diabetes, cardiovascular and cerebrovascular diseases, metabolic syndrome, estrogen-related malignant tumor and the like, and seriously affect the fertility, long-term health and life quality of the diseased females.
Intestinal flora is an indispensable "microbial organ" of our body and plays a vital role in maintaining health. In recent years, the link between intestinal microbiota and metabolic diseases has been the focus of attention of researchers who have found a correlation between intestinal health and polycystic ovary syndrome, and thus suggest that there may be some mechanisms in the intestinal microbiota that affect polycystic ovary syndrome.
Therefore, how to provide a microbial preparation capable of effectively improving PCOS, relieving related metabolic level imbalance and abnormal secretion of sex hormone, a female patient can try to use a health care product or a medicine containing specific probiotics, and bad life experience brought to the patient by the reproductive health problem caused by polycystic ovary syndrome is solved, so that new help can be provided for treating or assisting in treating PCOS based on intestinal flora as a target.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide lactobacillus reuteri for improving polycystic ovary syndrome and application thereof, in particular to lactobacillus reuteri Lactobacillus reuteri LR strain, a culture containing the lactobacillus reuteri Lactobacillus reuteri LR strain, a probiotic containing the lactobacillus reuteri and application of the lactobacillus reuteri in preparation of medicines or health care products with the function of preventing, relieving or treating polycystic ovary syndrome.
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 reuteri for improving polycystic ovary syndrome, which is named as lactobacillus reuteri Lactobacillus reuteri LR strain (homonymous: lactobacillus reuteri Limosilactobacillus reuteri), and has a preservation number of CGMCC No.24408 and a preservation date of 2022, 02 and 21 days.
The invention separates and stores a new lactobacillus reuteri which can improve polycystic ovary syndrome from healthy human breast milk samples in Suzhou, jiangsu province, and names the new lactobacillus reuteri Lactobacillus reuteri LR strain which can effectively improve polycystic ovary syndrome and related metabolic level disorder and symptoms of abnormal sex hormone level, and the specific expression is as follows: (1) Significantly improving ovarian tissue pathological symptoms of polycystic ovarian syndrome patients, including cystic follicles, corpus luteum quantity, testosterone level and the like; (2) Short chain fatty acid with rich antibacterial activity can be produced to participate in the immune reaction and endocrine regulation of the organism, so that the host metabolism level and sex hormone secretion are regulated; (3) can significantly improve the diversity of intestinal flora. Therefore, the lactobacillus reuteri Lactobacillus reuteri LR strain can be used for preparing products (such as medicines and the like) for preventing, relieving or treating polycystic ovary syndrome.
In addition, lactobacillus reuteri is a probiotic, so that the lactobacillus reuteri LR38 obtained by screening is high in safety when being used in products (such as medicines and the like) for preventing, relieving or treating polycystic ovary syndrome; and the traditional Chinese medicine composition does not generate drug resistance, improves the occurrence of adverse reactions of physiological and psychological aspects and the like caused by the administration of antibiotics of the patients with the conventional polycystic ovary syndrome, and can be used for preventing, relieving or treating the products of the female polycystic ovary syndrome for a long time.
The screening steps of the lactobacillus reuteri related by the invention are as follows:
(1) Selecting a sample of healthy human breast milk from Suzhou city of Jiangsu province, performing 10-time gradient dilution with physiological saline with the mass concentration of 0.9%, diluting for 3 times, coating on a solid culture medium, culturing at 37 ℃ for 48 hours, picking up 3 bacterial colonies with different forms, marking and purifying on the surface of an improved MRS solid culture medium, picking up single bacterial colonies, performing expanded culture at 37 ℃ with a liquid culture medium, and preserving with glycerol with the mass concentration of 40%.
(2) And (3) carrying out in-vitro physiological characteristic test on the preserved 3 single strains, screening out a single strain with better gastric acid and bile salt resistance (artificial simulation) and SCFA (short chain fatty acid) yield, and identifying the single strain.
In a second aspect, the present invention provides a culture of lactobacillus reuteri for ameliorating polycystic ovary syndrome, the culture comprising: inoculating lactobacillus reuteri Lactobacillus reuteri LR strain according to the first aspect into culture medium, and culturing at 30-38deg.C (such as 30deg.C, 32deg.C, 35deg.C, 37deg.C, 38deg.C, etc.) for 18-24 hr (such as 18 hr, 19 hr, 20 hr, 21 hr, 22 hr, 23 hr, 24 hr, etc.).
Other specific point values within the above numerical ranges are all selectable, and will not be described in detail herein.
Preferably, the formulation of the culture medium comprises: peptone, beef extract, glucose, lactose, yeast extract, diammonium hydrogen citrate, K 2 PO 4 ·3H 2 O、MgSO 4 ·7H 2 O、MnSO 4 L-cysteine.
Specifically, the formula composition of the culture medium (liquid) comprises: 10g of peptone, 10g of beef extract, 20g of glucose, 10g of lactose, 5g of yeast extract, 2g of diammonium hydrogen citrate and K 2 PO 4 ·3H 2 O 2g、MgSO 4 ·7H 2 O 0.6g、MnSO 4 0.01g, L-cysteine 1g.
The present invention preferably uses the above-described culture conditions, and lactobacillus reuteri Lactobacillus reuteri LR strain can achieve a stable growth period and has more excellent carbon source (glucose, fructose, lactose, galactose, maltose, sucrose, etc.) utilization ability.
In a third aspect, the invention provides a probiotic with a function of preventing, alleviating or treating polycystic ovary syndrome, which is characterized in that the probiotic with a function of preventing, alleviating or treating polycystic ovary syndrome comprises the lactobacillus reuteri Lactobacillus reuteri LR strain according to the first aspect.
The lactobacillus reuteri Lactobacillus reuteri LR strain related to the invention can be applied to related products alone or in combination with other strains.
Preferably, in the probiotic agent, the viable count of the lactobacillus reuteri Lactobacillus reuteri LR 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., and other specific point values within the numerical range may be selected, and will not be described in detail herein.
Preferably, the formulation of the probiotic agent 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, granules, solutions and other dosage forms.
Preferably, the probiotic agent 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 stachyose, fructooligosaccharide, galactooligosaccharide, mannooligosaccharide, trehalose, soybean oligosaccharide, resistant dextrin, xylooligosaccharide, polydextrose, alpha-lactalbumin or lactoferrin.
Further preferably, the probiotic agent further comprises lactobacillus acidophilus Lactobacillus acidophilus LA strain; the preservation number of the lactobacillus acidophilus Lactobacillus acidophilus LA strain is CGMCC No.24109 and 2021, 12 months and 15 days.
The invention also creatively discovers that the lactobacillus reuteri Lactobacillus reuteri LR strain can be compounded with the lactobacillus acidophilus Lactobacillus acidophilus LA strain for use in preventing, relieving or treating polycystic ovary syndrome, has an effect remarkably superior to that of a single microbial inoculum or other compound formulas, and shows that the LR38 strain and the LA88 strain have a synergistic effect in the aspects of regulating metabolic level, abnormal sex hormone secretion and other symptoms of a polycystic ovary syndrome patient, improving intestinal flora abundance of the patient and the like.
Preferably, the ratio of the viable count of lactobacillus reuteri Lactobacillus reuteri LR strain to lactobacillus acidophilus Lactobacillus acidophilus LA strain is (2-5): 1, e.g., 2:1, 3:1, 4:1, 5:1, etc., and other specific values within the numerical range are selectable, which will not be described in detail herein.
In the composite probiotic, the two strains have better synergistic effect when meeting the specific mass proportion relation.
In a fourth aspect, the invention provides the use of a lactobacillus reuteri Lactobacillus reuteri LR 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 or health care product having the function of preventing, alleviating or treating polycystic ovary syndrome.
Compared with the prior art, the invention has the following beneficial effects:
the invention separates and stores a new lactobacillus reuteri which can improve polycystic ovary syndrome, and names the lactobacillus reuteri Lactobacillus reuteri LR strain which can effectively improve polycystic ovary syndrome and the related metabolic level disorder and sex hormone level abnormality symptoms, and the specific appearance is: (1) Significantly improving ovarian tissue pathological symptoms of polycystic ovarian syndrome patients, including cystic follicles, corpus luteum quantity, testosterone level and the like; (2) Short chain fatty acid with rich antibacterial activity can be produced to participate in the immune reaction and endocrine regulation of the organism, so that the host metabolism level and sex hormone secretion are regulated; (3) can significantly improve the diversity of intestinal flora. Therefore, the lactobacillus reuteri Lactobacillus reuteri LR strain can be used for preparing products (such as medicines and the like) for preventing, improving or treating polycystic ovary syndrome.
The invention also creatively discovers that the lactobacillus reuteri Lactobacillus reuteri LR strain can be compounded with the lactobacillus acidophilus Lactobacillus acidophilus LA strain for use in preventing, improving or treating female polycystic ovary syndrome, has an effect remarkably superior to that of a single microbial inoculum or other compound formulas, and shows that the LR38 strain and the LA88 strain have a synergistic effect in the aspects of regulating metabolic symptoms, abnormal sex hormone secretion and other symptoms of a polycystic ovary syndrome patient, improving intestinal flora abundance of the patient and the like.
Drawings
FIG. 1 is a graph of the results of the detection of alpha diversity and differential flora by the intervention of Lactobacillus reuteri LR38 strain and/or Lactobacillus acidophilus LA88 strain.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The lactobacillus reuteri is lactobacillus reuteri Lactobacillus reuteri LR strain, the preservation unit is China general microbiological culture Collection center (CGMCC) No.24408, the preservation date is 2022, 02 month and 21 days, and the preservation address is Beicheng Chaoyang area Beicheng Xili No. 1 and 3.
The lactobacillus acidophilus is lactobacillus acidophilus Lactobacillus acidophilus LA strain, the preservation unit is China general microbiological culture Collection center, the preservation number is CGMCC No.24109, the preservation date is 2021, 12 and 15, and the preservation address is North Chen West Lu No. 1 of the Korean region of Beijing city.
Lactobacillus reuteri LR38 suspension referred to in the following: inoculating lactobacillus reuteri in MRS liquid culture medium, culturing at 37deg.C for 24 hr 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 24 hours to obtain bacterial solution; centrifuging the bacterial liquid at 8000g for 10min, and collecting supernatant, and filtering with 0.22 μm sterile filter membrane to obtain lactobacillus reuteri supernatant; and (5) re-suspending the thalli by using PBS to obtain the microbial inoculum.
The lactobacillus acidophilus LA88 suspension referred to in the following: inoculating lactobacillus acidophilus LA88 into MRS liquid culture medium, culturing at 37deg.C for 24 hr 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 24 hours to obtain bacterial solution; centrifuging the bacterial liquid at 8000g for 10min, and collecting supernatant, and filtering with 0.22 μm aseptic filter membrane to obtain lactobacillus acidophilus supernatant; and (5) re-suspending the thalli by using PBS to obtain the microbial inoculum.
The formulation composition of the MRS medium involved in the following includes: 10g of peptone, 10g of beef extract, 20g of glucose, 10g of lactose, 5g of yeast extract, 2g of diammonium hydrogen citrate and K 2 PO 4 ·3H 2 O 2g、MgSO 4 ·7H 2 O 0.6g、MnSO 4 0.01g, L-cysteine 1g.
Example 1
In this example, a strain of lactobacillus reuteri for improving polycystic ovary syndrome was selected as follows:
(1) Selecting a sample of healthy human breast milk from Suzhou city of Jiangsu province, performing 10-time gradient dilution with physiological saline with the mass concentration of 0.9%, diluting for 3 times, coating on a solid culture medium, culturing at 37 ℃ for 48 hours, picking out bacterial colonies with different forms, performing streak purification on the surface of the MRS solid culture medium, picking out single colonies, performing expansion culture at 37 ℃ with a liquid culture medium, and preserving with glycerol with the mass concentration of 35%.
(2) In vitro physiological property tests are carried out on the preserved single bacteria, and the method is specifically as follows:
(2.1) acid and bile salt resistance test:
the main reagents are pepsin, trypsin, sodium taurocholate and CaCO 3 Etc. Adjusting pH of MRS culture medium to 3.0, sterilizing at 121deg.C for 15min, inoculating 2% inoculum size into activated two-generation liquid culture expanded culture, culturing at 37deg.C for 24 hr, and measuring absorbance change ΔOD during 24 hr 600 A value; adding 0.1% bovine bile salt into MRS culture medium, sterilizing at 121deg.C for 15min, inoculating 2% inoculum size into activated two-generation liquid culture, culturing at 37deg.C for 24 hr, and measuring change ΔOD of absorbance during 24 hr 600 The value is finally selected to be two delta OD 600 The next experiment was performed with 10 strains of relatively large value.
Acid resistance experiment: based on PBS buffer solution with pH=6.8, the pH value is adjusted to 3.0 by using 37% hydrochloric acid, the mixture is sterilized at 121 ℃ for 15min, then the activated two-generation liquid culture is inoculated according to 10% inoculum size, the culture is cultured at 37 ℃, and the sampling and the determination of the viable count are respectively carried out at 0min, 30min, 60min, 90min and 120 min.
Bile salt resistance experiment: the liquid culture after two generations of strain activation is inoculated into MRS culture media (0.1%, 0.2%, 0.3%, 0.5% and 2% of bile salts in the culture media) containing different bile salt concentrations in an inoculation amount of 2%, meanwhile, MRS culture media without bile salts are used as a control, the culture media are cultivated for 24 hours at the constant temperature of 37 ℃ and then sampled to determine the number of viable bacteria, and the superior strain with acid resistance and bile salt resistance is screened by combining the last experimental result.
(2.2) acid generating ability test:
the acid producing capacity of the strain was determined by titration. The strain preserved by the glycerol pipe is inoculated into MRS liquid culture medium according to 2% of inoculum size after being activated, the culture is carried out for 24 hours at the constant temperature of 37 ℃, 10mL of fermentation liquor of each strain is taken in 50mL of sterile water, 2-3 drops of phenolphthalein with the concentration of 1g/L are dripped as an indicator, and 0.1mol/L NaOH standard solution is used for titration, and each sample is parallel for 3 times when pink solution appears and does not fade after 30 seconds. The non-inoculated MRS liquid culture medium is used as a blank control, and the count is calculatedThe calculation formula is as follows: total acidity/(g.L) -1 )＝(V1-V2)×c×100×V0 -1 (V1 is the volume of NaOH solution consumed by the sample, mL, V2 is the volume of NaOH solution consumed by the blank, mL, V0 is the total volume of the diluent, mL, and c is the concentration of standard NaOH, mol/L).
By combining the screening experiments, a strain with the strongest acid production capacity, gastric acid and bile salt tolerance is selected.
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. The colonies were observed to be milky white and full dots. The strain in the logarithmic growth phase is selected, and is detected by an optical microscope, and is observed by microscopic examination after smear and gram staining: gram staining is positive, the strain is in a rod shape, and no spore or flagellum exists.
(2) 16S rRNA molecular biology identification:
taking out the strain preserved at-80 deg.C, inoculating into a centrifuge tube filled with 20mL MRS liquid culture medium according to 2% ratio, culturing at 37 deg.C for 24 hr, centrifuging at 6000rpm 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. The sequences obtained by sequencing were subjected to nucleic acid sequence alignment in GeneBank, and the results show that the strain is Lactobacillus reuteri.
SEQ ID No:1：
ATACATGCAGTCGTACGCACTGGCCCAACTGATTGATGGTGCTTGCACCTGATTGACGATGGATCACCAGTGAGTGGGGACGGGTGAGTAACACGTAGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAACAACAAAAGCCACATGGCTTTTGTTTGAAAGATGGCTTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCGTGAGAGTAACTGTTCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGOCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAGCTCGCGAGAGTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTAACGCCCAAAGTCGGTGGCCTAACCATTATGGAGGGAGCCGCCTAAGGCGGGACAGATGACTGGGGTGAGTCGTACAAG。
Based on the results of the 16S rRNA molecular biological identification and morphological identification of example 2, it was confirmed that the strain belongs to Lactobacillus reuteri, designated as Lactobacillus reuteri Lactobacillus reuteri LR strain 38.
Example 3
In this example, the culture conditions of lactobacillus reuteri Lactobacillus reuteri LR strain were optimized as follows:
inoculating Lactobacillus reuteri LR38 into MRS liquid culture medium, culturing at different temperatures of 10-50deg.C for 48 hr, and measuring OD of the culture solution by enzyme-labeling instrument at intervals 600 Numerical values. The results are shown in Table 1:
TABLE 1
| 2h | 4h | 8h | 12h | 20h | 24h | 30h | 40h | 48h | |
| 10℃ | 0.359 | 0.550 | 0.558 | 0.558 | 0.562 | 0.565 | 0.565 | 0.566 | 0.565 |
| 20℃ | 0.502 | 0.560 | 0.558 | 0.562 | 0.574 | 0.581 | 0.581 | 0.604 | 0.600 |
| 30℃ | 0.495 | 0.506 | 0.617 | 0.683 | 1.075 | 1.774 | 2.359 | 2.634 | 2.700 |
| 35℃ | 0.583 | 0.627 | 0.700 | 1.964 | 3.659 | 4.770 | 4.755 | 4.736 | 4.708 |
| 40℃ | 0.576 | 0.604 | 0.711 | 1.563 | 3.374 | 4.581 | 4.554 | 4.406 | 4.390 |
| 45℃ | 0.550 | 0.592 | 0.637 | 0.785 | 1.982 | 2.325 | 2.330 | 2.271 | 2.223 |
| 50℃ | 0.519 | 0.521 | 0.585 | 0.610 | 0.759 | 1.425 | 1.420 | 1.411 | 1.408 |
The result shows that LR38 can reach the growth stabilization period after 20-24 hours of culture at 35-38 ℃.
The carbon source utilization capacity of the test strain LR38 was determined by performing a sugar fermentation reaction on the test strain LR38 using an API 50CHL medium (basal medium consisting of API 50CH test strips of 48 fermentable carbohydrates) and an API 50CH test strip according to API bacterial identification criteria. The principle of the method is that the strain to be measured is used for preparing suspension, the suspension is inoculated in each test strip small tube, and after the culture, the carbon source tube which can be utilized can produce acid due to fermentation, and the pH value is reduced, so that the indicator changes color.
As a result, strain LR38 can use various sugar sources such as glucose, fructose, lactose, galactose, maltose, sucrose and the like as carbon sources.
Example 4
This example demonstrates the gastric acid and bile salt tolerance of lactobacillus reuteri Lactobacillus reuteri LR strain, as follows:
(1) Preparing artificial gastric juice and bile salt:
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.
10% ox gall salt: 10.0g of ox gall salt (Walker, national pharmaceutical group chemical reagent Co., ltd., shanghai, china) was weighed into 100mL of sterile water, and was sterilized by filtration through a 0.22 μm filter membrane.
MRS medium containing 0.1% bile salts: MRS medium (Beijing Soy Co., ltd.) was prepared according to the instructions, and after high-pressure steam sterilization (121 ℃ C., 15 min), a proper amount of 10% ox gall salt solution was added to a final concentration of 0.1%.
(2) Gastric acid resistance test:
1.0mL of Lactobacillus reuteri LR38 strain suspension (concentration 1×10) 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 mixed with 9.0mL of artificial gastric juice with pH of 2.0, 2.5 and 3.0, and then cultured by anaerobic standing at 37 ℃, sampled after the beginning (0 h) and the treatment for 3h, respectively, and 3 parallel. The viable count was determined by pour culture and the viability was calculated as follows:
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 2.
TABLE 2
| pH of artificial gastric juice | Viable count N0 (0 h) | Viable count N1 (3 h) | Survival (%) |
| 2.0 | (6.82±0.21)×10 8 | (5.34±0.25)×10 8 | 78.3 |
| 2.5 | (6.79±0.15)×10 8 | (6.14±0.21)×10 8 | 90.4 |
| 3.0 | (6.80±0.23)×10 8 | (6.44±0.29)×10 8 | 94.7 |
(3) Bile salt resistance test:
1.0mL of Lactobacillus reuteri LR38 strain suspension (concentration 1×10) 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, GB4789.35-2016, and is mixed with 9.0mL of MRS culture medium containing 0.1% of ox gall salt, and the mixture is subjected to anaerobic static culture 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 bile salt treatment for 3 hours; n0: viable count of 0 h. The test results are shown in Table 3.
TABLE 3 Table 3
| Viable count N0 (0 h) | Viable count N1 (3 h) | Survival (%) |
| (6.75±0.18)×10 8 | (5.643±0.24)×10 8 | 83.6 |
As can be seen from table 2 and table 3, lactobacillus reuteri LR38 of the present invention has good gastric acid resistance, and the survival rate can reach 78.3% when incubated for 3 hours in artificial gastric juice with pH of 2.0; incubating in artificial gastric juice with pH of 2.5 for 3 hours, wherein the survival rate can reach 90.4%; incubation for 3h in artificial gastric juice with pH of 3.0, the survival rate can reach 94.7%. The good gastric acid and bile salt tolerance creates conditions for the field planting of the compound in the gastrointestinal tract, improving the metabolism of the organism and the hormone secretion level, and preparing the product for preventing, improving or treating the polycystic ovary syndrome.
Example 5
Safety in use test of lactobacillus reuteri Lactobacillus reuteri LR strain:
according to the 2010 edition Chinese pharmacopoeia, 6 SPF-grade mice (weight about 20-25 g) were selected, and 0.6mL of fresh MRS medium was injected into the abdominal cavity of the mice to obtain LR38 culture solution (viable count about 2×10) 9 CFU/mouse) and the change in body weight of each mouse was measured daily, and physiological changes such as mental state, behavioral activity, etc. before and after injection were recorded. As a result, the weight of the mice increased slightly within one weekNo obvious poisoning symptoms occur, the behavior is normal, no death phenomenon and no adverse reaction occur, so that the lactobacillus reuteri LR38 can be considered to be high in safety, and belongs to normal non-toxic probiotics.
Example 6
This example demonstrates the effect of lactobacillus reuteri Lactobacillus reuteri LR strain on relief of symptoms of polycystic ovary syndrome in rats, as follows:
female SD (Sprague-Dawley) rats at 8 weeks of age were purchased from Shanghai laboratory animal center and have been approved by ethical Committee for Shanghai laboratory animal research center (ethical number: 2022032003). All animals were kept under specific pathogen-free barrier conditions, alternately cycled 12 hours light and 12 hours dark and provided feed and water. Animal experiments strictly follow the national institutes of health, guidelines for laboratory animal care and use.
(1) Establishment of polycystic ovary syndrome rat model: the polycystic ovary syndrome of rats was induced using letrozole (from Jiangsu Hengrui medicine Co., ltd.) according to methods commonly used in the art (ref: wang MX, yin Q, xu X.ARat Model of Polycystic Ovary Syndrome with Insulin Resistance Induced by Letrozole Combined with High Fat Diet. Med Sci Monit.2020May 25;26:e922136.DOI: 10.12659/MSM.922136.). The 48 rats were randomly divided into 6 groups: control group (CTL), untreated polycystic ovary syndrome model group (MC), polycystic ovary syndrome group treated with lactobacillus reuteri LR38 (LR 38), polycystic ovary syndrome group treated with lactobacillus acidophilus LA88 (LA 88), polycystic ovary syndrome group treated with lactobacillus reuteri LR38 and lactobacillus acidophilus LA88 in combination (lr38+la 88, viable count ratio 3:1), polycystic ovary syndrome group treated with lactobacillus reuteri ATCC53608 and lactobacillus acidophilus LA88 in combination (ATCC 53608+la88, viable count ratio 3:1). The intervention of each group of microbial agents is 4 multiplied by 10 9 CFU/day, 1 week adaptation period was performed prior to dosing. To evaluate the effect of probiotics on polycystic ovary syndrome, we induced polycystic ovary syndrome symptoms with letrozole (1 mg/kg,1% carboxymethyl cellulose lavage) for 21 consecutive days, CTL rats lavaged with 1% carboxymethylCellulose replaces letrozole. All rats were fed normal diet for 28 consecutive days, and body weight (results shown in Table 4, units: g) and diet consumption were recorded every 3 days.
(2) Inoculating the strains into MRS liquid culture medium respectively, culturing at 37deg.C for 24 hr for activation, and 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 24 hours to obtain bacterial solution; centrifuging the bacterial liquid at 6000g for 10min, and collecting supernatant, and filtering with 0.22 μm sterile filter membrane to obtain supernatant; the cells were resuspended in PBS to obtain each bacterial suspension.
(3) Serum analysis: rats were fasted at night and sacrificed on day 29. Blood samples were collected, stored at 20℃for 1 hour, and then centrifuged at 3000g for 15min to obtain serum.
Determination of metabolic levels: total Cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) were measured using a biochemical analyzer (Beckman Coulter, calif.) (the results are shown in Table 5).
Determination of sex hormone levels: testosterone, luteinizing Hormone (LH), estradiol (E2), progesterone, anti-mullerian hormone (AMH), gonadotropin releasing hormone (GnRH) were measured using an enzyme-linked immunosorbent assay kit according to the manufacturer's protocol (wuhan purity biotechnology company) (results shown in table 6).
(4) Oral Glucose Tolerance Test (OGTT): rats were fasted at night and were tested for oral glucose tolerance on day 26. After baseline testing, rats were perfused with gastric glucose (2.5 g/kg body weight, 50% dextrose solution). Blood glucose concentrations of 30, 60, 90 and 120min after administration were measured by tail vein blood sampling (results shown in Table 7, units: mmol/L) using blood glucose test paper (trade name: excellent Jin Rui blood glucose test paper, roche).
(5) Fecal sampling and short chain fatty acid detection: the colon of the mice was collected and the feces were rapidly frozen in sterile tubes and stored at-80 ℃. The short chain fatty acid content was analyzed using gas chromatography-mass spectrometry and total SCFAs levels in ileal content, cecal content, colonic content and faeces were measured (results shown in Table 8, units/. Mu.mol/g).
TABLE 4 Table 4
As can be seen from table 4, rats in the MC group grew rapidly and grew faster after 4 weeks of intervention compared to the CTL group. In the pre-probiotic intervention group, the weight growth trend of rats in the LR38 group is slowed down, and the overall growth speed is reduced compared with that of rats in the MC group; in addition, it was unexpectedly found that the weight loss effect was more pronounced in rats of the lr38+la88 group when the lactobacillus reuteri and lactobacillus acidophilus were combined for intervention, especially at week 4, the average body weight tended to approach that of the CTL group.
TABLE 5
As can be seen from Table 5, the Triglyceride (TG) and low density lipoprotein cholesterol (LDL-C) levels were higher in the MC group and the Total Cholesterol (TC) and high density lipoprotein cholesterol (HDL-C) levels were reduced in the contrast CTL group, indicating that letrozole successfully induced metabolic disorders in rats. However, when probiotics intervened, we found that LR38 group rats had decreased TG and LDL-C levels and recovered TC and HDL-C levels. In addition, we have unexpectedly found that in the LR38+LA88 group, the serum TC, HDL-C, TG, LDL-C levels in rats were substantially restored to normal levels, and the animals tended to become CTL groups. Overall, lactobacillus reuteri LR38 had excellent effect of improving symptoms of metabolic disorder in polycystic ovary syndrome rats.
TABLE 6
The data in table 6 reflect the endocrine status of the experimental rats. By analyzing the concentration of serum sex hormone, we find that the levels of testosterone, LH and GnRH of MC rats are obviously higher, the symptoms of polycystic ovary syndrome are presented, and meanwhile, the levels of progesterone, E2 and AMH are lower, so that the abnormal ovarian function of the MC rats is reflected. However, the level of androgenic testosterone in LR38 rats showed a trend to decrease and the level of estrogens increased with the intervention of probiotics, i.e. the symptoms of polycystic ovary syndrome were improved. In addition, we find that the compound probiotics group LR38+LA88 has more obvious effect of restoring the estrogen level of the experimental rats, so that the symptoms related to polycystic ovary syndrome are relieved.
TABLE 7
| Group of | 0min | 30min | 60min | 90min | 120min |
| CTL | 5.7 | 8.4 | 9.5 | 7.7 | 5.8 |
| MC | 5.6 | 12.5 | 14.4 | 9.8 | 8.6 |
| LR38 | 5.7 | 9.6 | 10.0 | 8.4 | 6.5 |
| LR38+LA88 | 5.8 | 8.7 | 9.5 | 8.0 | 6.1 |
| LA88 | 5.5 | 10.3 | 12.8 | 9.2 | 7.6 |
| ATCC53608+LA88 | 5.7 | 10.5 | 12.6 | 9.0 | 7.4 |
Table 7 reflects the change of blood glucose concentration of experimental rats in 2 hours, compared with the CTL group, the MC rats can not normally regulate blood glucose concentration due to the fact that the regulation and control of blood glucose metabolism of the MC rats are blocked, but in the LR38 rats, the blood glucose metabolism capacity is improved, and the normal blood glucose metabolism can be basically recovered, especially when LR38+LA88 is combined, the effect on regulation and control of blood glucose metabolism of the model rats is more obvious.
TABLE 8
| Group of | Cecum total SCFAs | Total SCFAs of the colon | Fecal total SCFAs |
| CTL | 200.4 | 70.5 | 99.3 |
| MC | 176.3 | 55.2 | 76.5 |
| LR38 | 227.6 | 84.9 | 165.9 |
| LR38+LA88 | 252.8 | 92.2 | 170.4 |
| LA88 | 189.6 | 80.6 | 133.6 |
| ATCC53608+LA88 | 190.5 | 81.1 | 140.2 |
As can be seen from the results in Table 8, compared with the CTL group, the MC group rat intestinal flora metabolite has reduced total SCFAs in cecum, colon and feces, i.e. polycystic ovary syndrome affects intestinal flora steady state to reduce SCFAs content, and thus metabolic disorders such as insulin resistance may be induced, and the experimental rats presented in Table 8 have impaired blood glucose regulation. However, under the intervention of probiotics, the detected quantity of short chain fatty acid in the intestinal tract and the excrement of the rats tested in the LR38 group is obviously increased, that is, the intervention of lactobacillus reuteri can improve the problem of dysbacteriosis of intestinal tract of polycystic ovary syndrome, and the content of SCFAs is increased, thereby being beneficial to the energy intake of organisms, the regulation of immunity, the improvement of metabolic level and the like. In addition, we found that this effect was more pronounced when LR38 and LA88 were combined.
Example 6
This example demonstrates the effect of lactobacillus reuteri Lactobacillus reuteri LR strain on rat ovarian tissue pathology:
(1) The PCOS rat model was constructed as described in example 5.
(2) Rat ovarian tissue pathology analysis:
immediately after euthanasia of the experimental rats, ovarian and colon samples were taken from CTL, MC, LR38 and lr38+la88 groups and fixed with 4% paraformaldehyde. Hematoxylin-eosin (HE) staining and immunohistochemistry were performed. Determination of morphology in the references (Wang MX, yin Q, xu X.A Rat Model of Polycystic Ovary Syndrome with Insulin Resistance Induced by Letrozole Combined with High Fat Diet. Med Sci Monit.2020May 25; 26:e922136.): the granulosa cell layer becomes weak and the follicle with the membrane cell layer thickened is considered to be a cystic follicle. At the same time, the number of vesicles, vesicle duty ratio (vesicle duty ratio=number of vesicles/total number of vesicles), and corpus luteum were calculated. The results are shown in Table 9.
TABLE 9
| Group of | Number of cystic follicles | Luteal digit (number) | Thickness of granulosa cell layer (μm) |
| CTL | 0.13 | 8.63 | 56.93 |
| MC | 8.88 | 2.63 | 22.67 |
| LR38 | 3.13 | 6.75 | 43.71 |
| LR38+LA88 | 2.13 | 8.13 | 48.62 |
In a normal letrozole-induced polycystic ovary syndrome rat model, the pathological state of the ovary is mainly caused by the disturbance of sex hormone level, and the ovary presents symptoms such as luteal phase reduction, polycystic vesicles and the like. Compared with the CTL group, the number of cystic follicles of the rats in the MC group is obviously increased along with the rapid decrease of the corpus luteum number, the thickness of the cell layers is thinned, but the probiotic dry prognosis, the LR38 group rats can effectively reduce the weight index of the ovaries and the number of the cystic follicles of the rats, and simultaneously partially restore the corpus luteum number of the ovaries. In addition, we have unexpectedly found that the ovary morphology of the LR08+LA88 combined rat is basically recovered to be normal, the corpus luteum is rich, the follicles are more, and the mice basically tend to be in CTL groups. In other words, lactobacillus reuteri LR38 is able to restore morphological abnormalities of the ovaries by modulating sex hormone levels.
Example 7
This example demonstrates that lactobacillus reuteri LR38 regulates intestinal flora function as follows:
(1) The PCOS rat model was constructed as described in example 5.
(2) Intestinal flora analysis: feces were collected from rats in CTL, MC, LR38 and lr38+la88 groups. Total DNA was extracted from 200mg of stool using QIAamp stool DNA extraction kit (Qiagen). The 16S rRNA V3-V4 region was PCR amplified using 341F (SEQ ID No:2:5 '-CCTACGGGNGGCWGCAG-3') and 805R (SEQ ID No:3:5 '-GACTACHVGGGTATCTAATCC-3') primers. The final 16S rRNA gene amplicon library was sequenced on the Miseq platform (Illumina) using a 2X 300bp paired-end protocol. The following analysis was performed using Usearch11 to combine paired end reads obtained and retain reads of length 400 pb. All mass filter sequences were mapped to ASVs without chimeras and ASV abundance tables were created using userch 11 with default settings. The 16S rRNA database of RDP training set (v 18 version) was used as the reference database. Based on ASVs abundance tables, index of Chao1 and Shannon diversity (alpha diversity) was calculated using userch, intervention effects were determined by one-way analysis of variance (ANOVA), and differences between groups were analyzed post-hoc by Tukey test significance difference test. The results of the lactobacillus reuteri LR38 intervention on alpha diversity detection are shown in figure 1.
As can be seen from fig. 1, the microbial community α diversity was significantly reduced in the MC group rats compared to the CLT group. Specifically, the microbial abundance decreases (expressed as the Chao1 index), and the microbial diversity decreases (expressed as the Shannon index). Comparison of LR38 group with MC group shows that LR38 increases α diversity of the microbiota and that the probiotic composite formulation has more pronounced effect on improving intestinal flora diversity in polycystic ovary syndrome rats when lactobacillus reuteri LR38 and lactobacillus acidophilus LR88 co-act.
The applicant states that the present invention is illustrated by the above examples as a lactobacillus reuteri for improving polycystic ovary syndrome and its use, but the present invention is not limited to, 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. The lactobacillus reuteri for improving polycystic ovary syndrome is named as lactobacillus reuteri Lactobacillus reuteri LR strain Lactobacillus reuteri LR, and has a preservation number of CGMCC No.24408 and a preservation date of 2022, 02 and 21.
2. A culture of lactobacillus reuteri for ameliorating polycystic ovary syndrome, the method of preparing the culture comprising: inoculating lactobacillus reuteri Lactobacillus reuteri LR strain according to claim 1 into culture medium, and culturing at 30-38deg.C for 18-24 hr.
3. A probiotic with a function of preventing, alleviating or treating polycystic ovary syndrome, characterized in that the probiotic with a function of preventing, alleviating or treating polycystic ovary syndrome comprises lactobacillus reuteri Lactobacillus reuteri LR strain according to claim 1.
4. The probiotic agent for preventing, alleviating or treating polycystic ovary syndrome according to claim 3, wherein the viable count of lactobacillus reuteri Lactobacillus reuteri LR strain in the probiotic agent is not less than 1 x 10 8 CFU/mL or 1X 10 8 CFU/g。
5. The probiotic having the effect of preventing, alleviating or treating polycystic ovary syndrome of claim 3, wherein said probiotic is in a dosage form comprising lyophilized powder, capsule, tablet or granule.
6. A probiotic with a prevention, alleviation or treatment of polycystic ovary syndrome according to claim 3, characterized in that said probiotic further comprises a protective agent and/or an auxiliary additive.
7. The probiotic with a prevention, alleviation or treatment of polycystic ovary syndrome of claim 6, wherein said protective agent comprises skim milk powder;
the auxiliary additive comprises any one or a combination of at least two of stachyose, fructooligosaccharide, galactooligosaccharide, mannooligosaccharide, trehalose, soybean oligosaccharide, resistant dextrin, xylooligosaccharide, polydextrose, alpha-lactalbumin or lactoferrin.
8. The probiotic having the effect of preventing, alleviating or treating polycystic ovary syndrome of claim 3, wherein said probiotic further comprises lactobacillus acidophilus Lactobacillus acidophilus LA strain; the preservation number of the lactobacillus acidophilus Lactobacillus acidophilus LA strain is CGMCC No.24109 and 2021, 12 months and 15 days.
9. The probiotic for preventing, alleviating or treating polycystic ovary syndrome of claim 8, wherein the ratio of the viable count of lactobacillus reuteri Lactobacillus reuteri LR strain to lactobacillus acidophilus Lactobacillus acidophilus LA strain is (2-5): 1.
10. Use of a lactobacillus reuteri Lactobacillus reuteri LR strain according to claim 1 or a culture according to claim 2 or a probiotic according to any of claims 3-9 in the manufacture of a medicament or health care product having the function of preventing, alleviating or treating polycystic ovary syndrome.
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| CN115364126A (en) * | 2021-05-17 | 2022-11-22 | 上海交通大学医学院附属仁济医院 | Application of lactobacillus reuteri in preparation of medicine for treating polycystic ovarian syndrome |
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| CN117305188A (en) * | 2023-11-27 | 2023-12-29 | 山东中微众康生物科技有限公司 | Lactobacillus acidophilus RZKLa0701 capable of promoting bone growth of children and application thereof |
| CN117305188B (en) * | 2023-11-27 | 2024-02-13 | 山东中微众康生物科技有限公司 | Lactobacillus acidophilus RZKLa0701 capable of promoting bone growth of children and application thereof |
| CN117844714A (en) * | 2024-03-04 | 2024-04-09 | 微康益生菌(苏州)股份有限公司 | Probiotics for regulating and controlling brain-derived neurotrophic factors and sex hormones and application thereof |
| CN117844714B (en) * | 2024-03-04 | 2024-05-17 | 微康益生菌(苏州)股份有限公司 | Probiotics for regulating and controlling brain-derived neurotrophic factors and sex hormones and application thereof |
| CN118667725A (en) * | 2024-08-22 | 2024-09-20 | 中国农业科学院北京畜牧兽医研究所 | Probiotic compound agent for improving fertility of female animals and application |
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