CN113317447A - Application of lactobacillus plantarum CCFM1019 in relieving polycystic ovarian syndrome - Google Patents
Application of lactobacillus plantarum CCFM1019 in relieving polycystic ovarian syndrome Download PDFInfo
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Abstract
The invention discloses application of lactobacillus plantarum CCFM1019 in relieving polycystic ovary syndrome, and belongs to the technical field of functional microorganisms. The invention provides a method for preparing fermented soybean milk by using lactobacillus plantarum CCFM1019, wherein the viable count of the soybean milk fermented by the lactobacillus plantarum CCFM1019 is 1 multiplied by 108Under the condition of CFU/mL, the medicine can improve the ovarian polycystic lesionAnd the abnormal level of sex hormones such as estradiol and anti-mullerian hormone caused by the polycystic ovary syndrome is regulated, and the polycystic ovary syndrome and related complications have certain relieving effect.
Description
Technical Field
The invention relates to application of lactobacillus plantarum CCFM1019 in relieving polycystic ovary syndrome, and belongs to the technical field of functional microorganisms.
Background
Polycystic ovary syndrome (polycystic ovary syndrome) is used as a common endocrine disease of females in adolescence and child bearing age, common clinical symptoms comprise obesity, acne, infrequent menstruation, hirsutism, endocrine dyscrasia, infertility and the like, and the common clinical symptoms greatly influence the physical and mental health of patients.
At present, the research on the pathogenesis of polycystic ovarian syndrome is in an exploration stage, and the drug therapy is still the first scheme for relieving the PCOS symptom, and the commonly used drugs comprise oral contraceptives, ovulation promoting drugs, insulin sensitizers and the like. These drugs regulate hormone levels, primarily by regulating the menstrual cycle, reducing insulin resistance, and promote ovulation. However, the use of the above drugs also has significant side effects, such as the possibility of increasing the risk of insulin resistance by oral contraceptives, the possibility of increasing the risk of bladder cancer by insulin sensitizers, and the like. Therefore, there is a need to find a safer and more effective means of PCOS mitigation.
The study that the intestinal flora participates in the pathogenesis of polycystic ovarian syndrome (intestinal flora imbalance-brain central signal receiving-gonadotropin and sex hormone abnormal secretion) through the intestinal-brain axis provides a new idea for treating polycystic ovarian syndrome. Lactobacillus plantarum CCFM1019 is a reported probiotic with relief of polycystic ovary syndrome. The strain is 1 × 109The number of CFU's acting on rats, or 1X 108The number of CFU's acting on mice helps to alleviate polycystic ovarian syndromeCombined symptoms, but less relief is apparent at doses below the above-mentioned doses.
Disclosure of Invention
The invention aims to provide lactobacillus plantarum CCFM1019 fermented soybean milk with a function of relieving polycystic ovarian syndrome and application thereof.
A first object of the present invention is to provide a method for preparing fermented soybean milk, comprising the steps of:
s1, mixing and soaking soybeans and water according to the mass ratio of 1 (10-12);
s2, grinding the soybeans soaked in the S1 into pulp, filtering the pulp by using 120-mesh filter cloth, boiling the pulp at 100 ℃ for at least 5min, and mixing the pulp with sugar;
s3, inoculating lactobacillus plantarum CCFM1019 into the slurry after S2 size mixing, standing and fermenting at 30-37 ℃ to enable the concentration of the lactobacillus plantarum CCFM1019 to reach at least 1 x 108CFU/mL。
In one embodiment, lactobacillus plantarum CCFM1019, deposited as GDMCC No.60333, is disclosed in patent document CN 108384735B.
In one embodiment, the method comprises the steps of:
(1) selecting raw materials: selecting soybeans with no mildew, rot and deterioration on the surface;
(2) raw material treatment: the soybeans selected in the step (1) are mixed according to the weight ratio of 100g of soybeans: soaking in 1L water for one night;
(3) preparing soybean milk: grinding the soybeans soaked in the step (2) for 2min at 80 ℃ and 18000rpm by using a wall breaking machine, filtering the hot soybeans by using 120-mesh gauze, boiling the soybeans for 5min at 100 ℃, adding sucrose with the final concentration of 70g/L to adjust the sweetness of the soybean milk, sterilizing the soybean milk for 20min at 115 ℃, and cooling the soybean milk to room temperature.
(4) Fermenting soybean milk: inoculating Lactobacillus plantarum CCFM1019 into cooled soybean milk to make the concentration of the inoculated bacteria reach 5 x 106CFU/mL, standing at 37 deg.C for fermenting for 8 hr to obtain thallus concentration of 1 × 108CFU/mL fermented soybean milk.
The invention also provides the fermented soybean milk prepared by the method.
The invention also provides application of the fermented soybean milk in improving or assisting in treating the symptoms of polycystic ovarian syndrome.
The invention also provides application of the fermented soybean milk in improving or assisting in treating ovarian polycystic lesion caused by polycystic ovarian syndrome.
In one embodiment, the ameliorating or alleviating a symptom of polycystic ovary syndrome comprises ameliorating an abnormal level of estradiol, ameliorating an abnormal level of anti-mullerian hormone.
The invention also provides application of the fermented soybean milk in preparing functional food or functional food composition for improving or relieving polycystic ovarian syndrome.
The invention has the beneficial effects that: the invention provides a method for preparing fermented soybean milk by using lactobacillus plantarum CCFM1019, wherein the viable count of the soybean milk fermented by the lactobacillus plantarum CCFM1019 is 1 multiplied by 108Under the condition of CFU/mL, the traditional Chinese medicine composition can improve the ovarian polycystic lesion, regulate the abnormal level of sex hormone (estradiol and anti-mullerian hormone) caused by the polycystic ovarian syndrome, and has certain relieving effect on the polycystic ovarian syndrome and related complications thereof.
Drawings
FIG. 1 shows the viable count of fermented soybean milk with Lactobacillus plantarum CCFM 1019;
FIG. 2 shows the effect of Lactobacillus plantarum CCFM1019 fermented soymilk on the pathology of ovarian tissues in rats with polycystic ovarian syndrome;
FIG. 3 is a graph showing the effect of Lactobacillus plantarum CCFM1019 fermented soymilk on the estradiol levels in polycystic ovary syndrome rats;
figure 4 is the effect of lactobacillus plantarum CCFM1019 fermented soy milk on the anti-mullerian hormone levels in rats with polycystic ovarian syndrome: significant difference compared to model group, P < 0.05; # has a significant difference compared to the blank group, P <0.05
Detailed Description
The lactobacillus plantarum LP4 and the lactobacillus plantarum LP5 mentioned in the following examples are commonly used lactobacillus plantarum in two fermented drinks.
Example 1: preparation of lactobacillus plantarum CCFM1019 fermented soybean milk
The fermented soybean milk is prepared by the following method:
(1) selecting raw materials: selecting soybeans with no mildew, rot and deterioration on the surface;
(2) raw material treatment: soaking the soybeans selected in the step (1) in water overnight (the soybean water ratio is 100 g: 1L);
(3) preparing soybean milk: grinding the soybeans soaked in the step (2) by using a wall breaking machine (grinding conditions are 80 ℃, 2min and 18000rpm), filtering the ground soybeans with 120-mesh gauze when the ground soybeans are hot, boiling the ground soybeans for 5min at 100 ℃, adding cane sugar for size mixing (the ratio of cane sugar to soya-bean milk is 7 g: 100mL), sterilizing the ground soybeans for 20min at 115 ℃, and cooling the ground soybeans to room temperature.
(4) Fermenting soybean milk: inoculating Lactobacillus plantarum CCFM1019 into cooled soybean milk to make the concentration of the inoculated bacteria reach 5 x 106CFU/mL, standing at 37 deg.C for fermenting for 8 hr to obtain thallus concentration of 1 × 108CFU/mL fermented soybean milk.
The inventors also tried to prolong the fermentation time, and the concentration reached 1X 10 under the soybean milk fermentation environment8CFU/mL is basically in a state of the saturation of the number of thalli, and the problems of reduction of the activity of lactobacillus plantarum cells and the like easily occur when the fermentation time is continuously prolonged, so that the action effect is influenced.
Example 2 Lactobacillus plantarum CCFM1019 fermented soymilk sustained polycystic ovarian syndrome rat ovarian polycystic lesion
After the rats were acclimated for 1 week, 36 SD female rats were randomly divided into a blank group, a model group, a Danying-35 group, a CCFM1019 fermented soymilk group, a Lactobacillus plantarum LP4 fermented soymilk group, and a Lactobacillus plantarum LP5 fermented soymilk group, each group consisting of 6 rats. In the molding period (3 weeks), the blank group was gavaged with 1% CMC solution every day, the final dose reached 0.5mL/100g BW, the model group, the Daying-35 group and the lactic acid bacteria fermented soybean milk group were gavaged with letrozole suspension (20 letrozole suspended in 250mL 1% CMC solution) every day, and the final gavage volume reached 0.5mL/100g BW. Expected dryness (4 weeks), 2mL of unfermented soymilk in the blank group and the model group, respectively, and darying-35 group in the suspension (1 tablet darying-35 suspended in 50mL of 1% CMC solution) in the stomach per day, the final stomach filling volume reaches 0.45mL/100g BW, and the lactobacillus fermented soymilk in the group is 2mL of fermented soymilk in the stomach.
The grouping and treatment method of experimental animals is shown in table 1:
TABLE 1 grouping and handling of laboratory animals
At the end of the test, one side of the ovary of the rat is taken to prepare a paraffin section, and after HE staining, the tissue morphology is observed under a light mirror and photographed for pathological evaluation. The method comprises the following specific steps:
(1) fixing: the tissue sample is washed by normal saline and immediately put into a neutral paraformaldehyde fixing solution (4%) for fixing, and the fixing time is generally within 72 h.
(2) Washing: rinsing or soaking with running water for several hours or overnight.
(3) And (3) dehydrating: the sample is dehydrated by 70%, 80% and 90% ethanol solutions for 30min, respectively, and then placed for 1 time at 95% for 20min and 2 times at 100% for 10min each time.
(4) And (3) transparency: 1/2 pure alcohol and 1/2 xylene mixed solution 10min, xylene I10 min, and xylene II 10min (until transparent).
(5) Wax dipping: the sample was placed in paraffin (62 ℃ C.) for 2 h.
(6) Embedding: the largest surface is positioned on the bottom layer, so that the cut surface texture surface occupies the largest area.
(7) Slicing: the wax pieces were cut into 5 μm thick sections with a manual microtome.
(8) Spreading and sticking (fishing out pieces): the water bath was opened to maintain the water temperature at 42 ℃ and the slices were spread flat on the water surface.
(9) Baking slices: the slide along with the slide rack was placed in a 55 ℃ dry box for about 2 hours until the wax melted.
(10) Hydration: paraffin sections are dewaxed for 10min respectively by dimethylbenzene I and II, then put into alcohol solutions of 100%, 95%, 90%, 80% and 70% for 5min respectively, and then put into distilled water for 3 min.
(11) Primary dyeing: the sections were stained in hematoxylin for about 20 s.
(12) Washing with water: rinsing with running water for about 15 min. The color of the slices is changed to blue, but the flowing water is not too large to prevent the slices from falling off.
(13) Differentiation: the slices were placed in 1% ethanol hydrochloride solution for 7s to fade. The color of the slices turns red and is lighter.
(14) Rinsing: the slices are washed in tap water for 15-20min to restore blue color.
(15) Counterdyeing: immersing in eosin dye solution, and immediately taking out for dewatering.
(16) And (3) dehydrating: the slices are sequentially processed by 95% ethanol I, 95% ethanol II and 70% ethanol, and then added with 80% ethanol for 50s and absolute ethanol for 2 min.
(17) And (3) transparency: the slices were placed in 1/2 absolute ethanol, 1/2 xylene for 1min, 2min each in xylene I and II.
(18) Sealing: after the slices are xylene transparent, the gum can be diluted with xylene to a suitable consistency using neutral gum as the occlusal agent.
The experimental results are shown in fig. 2: the ovary surface of the blank group of rats is ruddy in color, the corpus luteum is visible, and the cystic follicle is absent, while the ovary surface of the model group of rats is lightened in color, the corpus luteum number is reduced, and the cystic follicle number is increased. The lactobacillus plantarum CCFM1019 fermented soybean milk for intragastric administration can obviously improve the ovarian lesion of a rat with polycystic ovarian syndrome, and has more obvious effect compared with other two lactobacillus plantarum fermented soybean milk groups, which shows that the lactobacillus plantarum CCFM1019 fermented soybean milk can obviously improve the polycystic lesion caused by the polycystic ovarian syndrome.
Example 3 Lactobacillus plantarum CCFM1019 fermented soymilk restores the estradiol levels in rats with polycystic ovarian syndrome
Animal experiment test grouping and treatment are the same as example 2. At the end of the test, rats are fasted for 12 hours without water prohibition, and blood is collected from the heart after anesthesia by intraperitoneal injection of 0.5mL/10g of 1% sodium pentobarbital solution. Centrifuging blood sample at 3000 Xg and 4 deg.C for 10min, collecting supernatant, and determining estradiol content in blood serum according to the detection method of the kit.
The experimental result is shown in fig. 3, compared with the model group, the content of estradiol in the serum of the rat intragastrically perfused with the lactobacillus plantarum CCFM1019 fermented soybean milk is increased from 21.87 +/-1.506 pg/mL to 26.87 +/-3.332 pg/mL compared with the model group, is close to 25.16 +/-2.744 pg/mL of the blank group, and the effect is obviously better than that of the other two strains of lactobacillus plantarum fermented soybean milk, which indicates that the lactobacillus plantarum CCFM1019 fermented soybean milk can increase the level of estradiol in the rat with polycystic ovary syndrome.
Example 4 Lactobacillus plantarum CCFM1019 fermented soymilk reduces the anti-Mullerian hormone levels in rats with polycystic ovarian syndrome
Animal experiment test grouping and treatment are the same as example 2. At the end of the test, rats are fasted for 12 hours without water prohibition, and blood is collected from the heart after anesthesia by intraperitoneal injection of 0.5mL/10g of 1% sodium pentobarbital solution. Centrifuging blood sample at 3000 Xg and 4 deg.C for 10min, collecting supernatant, and determining the content of anti-Mullerian hormone in blood serum according to the detection method of kit.
The experimental results are shown in figure 4, and compared with the blank group, the anti-mullerian hormone level in the serum of the model group rats is obviously increased to 4.53 +/-1.007 ng/L, which indicates that the polycystic ovary syndrome causes the anti-mullerian hormone level to be increased; compared with the model group, the level of anti-mullerian hormone in serum of rats intragastrically perfused with the lactobacillus plantarum CCFM1019 fermented soybean milk is remarkably reduced to 3.06 +/-0.1812 ng/L, which is close to the level of the blank group, and the fact that the lactobacillus plantarum CCFM1019 fermented soybean milk can reduce the level of anti-mullerian hormone in the rat with polycystic ovarian syndrome is shown.
Comparative example 1:
the specific implementation manner is the same as that in example 3, except that lactobacillus plantarum CCFM1019 fermented soybean milk is replaced by lactobacillus plantarum CCFM1019 bacterial suspension re-suspended in fresh soybean milk (the final concentration of lactobacillus plantarum CCFM1019 is 1X 10)8CFU/mL, unfermented), and suspension of Lactobacillus plantarum CCFM1019 suspended in sterile physiological saline (final concentration of Lactobacillus plantarum CCFM1019 is 1X 10)8CFU/mL), respectively measuring the content of estradiol in serum of rats with polycystic ovary syndrome, and the results show that the content of estradiol in serum of rats in a lactobacillus plantarum CCFM1019 soybean milk suspension group is 22.01 +/-1.676 pg/mL, the content of estradiol in serum of rats in a physiological saline suspension group is 22.87 +/-2.126 pg/mL, and the level of estradiol in rats after gastric lavage of the lactobacillus plantarum CCFM1019 soybean milk suspension or the physiological saline suspension is not obviously changed compared with that in a model group (21.87 +/-1.506 pg/mL), and the results are shown in the tableThe ratio of Lactobacillus plantarum CCFM1019 to 1 × 108The number of CFUs acted on the rats and had no significant effect on estradiol levels.
Comparative example 2:
the specific implementation manner is the same as that in example 4, except that lactobacillus plantarum CCFM1019 fermented soybean milk is replaced by lactobacillus plantarum CCFM1019 bacterial suspension resuspended in fresh soybean milk (the final concentration of lactobacillus plantarum CCFM1019 is 1X 10)8CFU/mL, unfermented), and suspension of Lactobacillus plantarum CCFM1019 suspended in sterile physiological saline (final concentration of Lactobacillus plantarum CCFM1019 is 1X 10)8CFU/mL), respectively measuring the content of serum anti-Mullerian hormone of a rat with polycystic ovarian syndrome, and the results show that the serum anti-Mullerian hormone level of a model group is 4.53 +/-1.007 ng/L, the serum anti-Mullerian hormone level of a rat with lactobacillus plantarum CCFM1019 soybean milk suspension is 4.46 +/-0.9514 ng/L, and the serum anti-Mullerian hormone level of a rat with physiological saline suspension is 4.39 +/-1.268 ng/L, and the results show that the content of the serum anti-Mullerian hormone of the rat with the polycystic ovarian syndrome is 1 multiplied by 10 CCFM10198The number of CFUs acted on the rats and the effect of anti-mullerian hormone was not significant.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A preparation method of fermented soybean milk is characterized by comprising the following steps:
s1, mixing and soaking soybeans and water according to the mass ratio of 1 (10-12);
s2, grinding the soybeans soaked in the S1 into thick liquid, filtering, boiling the thick liquid for at least 5min at 90-100 ℃, and mixing the thick liquid with sugar;
s3, inoculating lactobacillus plantarum CCFM1019 into the slurry after S2 size mixing, standing and fermenting at 30-37 ℃ to enable the concentration of the lactobacillus plantarum CCFM1019 to reach at least 1 x 108CFU/mL。
2. The method as claimed in claim 1, wherein the filtration of S2 is a 100 mesh filter cloth.
3. The method of claim 1, wherein the S3 strain is further sterilized prior to inoculation.
4. The method according to claim 3, wherein the sterilization is performed at 115-121 ℃ for 15-20 min.
5. The method according to any one of claims 1 to 4, wherein the Lactobacillus plantarum CCFM1019 has a accession number GDMCC No. 60333.
6. A method according to any one of claims 1 to 5, comprising the steps of:
(1) selecting raw materials: selecting soybeans with no mildew, rot and deterioration on the surface;
(2) raw material treatment: the soybeans selected in the step (1) are mixed according to the weight ratio of 100g of soybeans: soaking in 1L water for one night;
(3) preparing soybean milk: grinding the soaked soybeans in the step (2) at 80 ℃, 18000rpm for 2min, filtering with 120-mesh gauze, boiling the pulp at 100 ℃ for 5min, adding sucrose with the final concentration of 70g/L to adjust the sweetness of the soybean milk, sterilizing at 115 ℃ for 20min, and cooling to room temperature.
(4) Fermenting soybean milk: inoculating Lactobacillus plantarum CCFM1019 into cooled soybean milk to make the concentration of the inoculated bacteria reach 5 x 106CFU/mL, standing at 37 deg.C for fermenting for 8 hr to obtain thallus concentration of 1 × 108CFU/mL fermented soybean milk.
7. Fermented soy milk prepared by the method according to any of claims 1 to 6.
8. Use of fermented soy milk according to claim 7 for ameliorating or adjunctively treating the symptoms of polycystic ovary syndrome.
9. The use as claimed in claim 8, wherein the amelioration or alleviation of symptoms of polycystic ovary syndrome comprises amelioration of abnormal estradiol levels and/or amelioration of abnormal anti-mullerian hormone levels.
10. Use of the fermented soybean milk of claim 7 for producing a functional food or a functional food composition for ameliorating or alleviating polycystic ovary syndrome.
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