CN110960561B - Probiotic composition for regulating balance of gynecological flora - Google Patents
Probiotic composition for regulating balance of gynecological flora Download PDFInfo
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- CN110960561B CN110960561B CN201911418368.9A CN201911418368A CN110960561B CN 110960561 B CN110960561 B CN 110960561B CN 201911418368 A CN201911418368 A CN 201911418368A CN 110960561 B CN110960561 B CN 110960561B
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C11/00—Milk substitutes, e.g. coffee whitener compositions
- A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
- A23C11/06—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing non-milk proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/125—Casei
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- Molecular Biology (AREA)
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- Epidemiology (AREA)
- Gynecology & Obstetrics (AREA)
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Abstract
The invention discloses a composite microbial inoculum for adjusting the balance of gynecological flora, which comprises the following components: lactobacillus crispatus, lactobacillus casei LC-N235 and other acceptable pharmaceutical excipients. The Lactobacillus crispatus is preferably Lactobacillus crispatus CGMCC 12743. The lactobacillus casei LC-N235 is preserved in China general microbiological culture collection center with the preservation number of CGMCC 18057. The compound microbial inoculum can be prepared into oral liquid or fermented food, preferably fermented milk, fermented soymilk and the like.
Description
Technical Field
The invention relates to a probiotic composition for regulating balance of gynecological flora, belonging to the technical field of microorganisms.
Background
A large amount of bacteria live in the gastrointestinal tract of a normal human, after the newborn is born for 2 hours, different bacteria enter the human body, a normal flora is gradually established, about 500 kinds of bacteria belong to more than 50 kinds of bacteria in the adult human body, and the total number of the bacteria exceeds 100 trillion. The intestinal bacterial flora is an extremely complex ecosystem of aerobic, anaerobic and facultative anaerobic bacteria, which normally reproduce in the host without causing disease and have positive effects on the health of the organism, including aiding digestion, absorption, nutrition, immunity and antagonism of pathogenic bacteria. The probiotic bacteria refer to bacteria with physiological activity beneficial to human body, such as Bifidobacterium, Lactobacillus, yeast, etc. The probiotic preparation used in clinic comprises various solid (capsules and tablets) or liquid (oral liquid and fermented milk) dosage forms, and can be divided into single-bacterium preparations and multi-viable-bacterium preparations according to different strains.
According to statistics, bacterial vaginitis is easy to occur to women in the childbearing period, various vaginitis discovered clinically is often mixed infection, symptoms such as increased leucorrhea, pruritus vulvae rash, vaginal burning pain and the like exist after diseases, and serious influence is brought to physical and mental health and work and study of the women. The lactobacillus in the vagina of the patient with the bacterial vaginitis is obviously reduced and even disappears. Although clinically common antibiotic treatment can be temporarily controlled, the refractory recurrence rate is high.
Although direct vaginal administration has good effect, the vaginal administration is inconvenient, and many women have conflicting psychology. The prior art has demonstrated that orally administered lactic acid bacteria partially enter the vagina, but with low efficiency. The invention aims to provide a probiotic composition for regulating the balance of gynecological flora, in particular to a novel food-derived safe probiotic introduced, which can directionally increase lactobacillus entering vagina through intestinal tract, synergistically enhance the effect of treating bacterial vaginitis and help to restore the balance of vaginal flora.
Disclosure of Invention
The invention aims to provide a probiotic composition for regulating the balance of gynecological flora, in particular to a probiotic composition which can synergistically enhance the effect of treating bacterial vaginitis by introducing new safe probiotics from food through at least directionally increasing lactobacillus entering vagina and is beneficial to restoring the balance of vaginal flora.
The vaginal flora of healthy women is mainly composed of lactobacillus, including lactobacillus crispatus, lactobacillus jensenii, lactobacillus gasseri and the like. Normally lactobacilli protect the vagina, while disturbances in vaginal microecology dominated by lactobacilli can lead to vaginitis. Clinical application proves that the lactobacillus crispatus has the best effect.
Bacterial Vaginosis (BV) is the most common genital infectious disease of women of childbearing age and is characterized in that the balance of flora inhabiting in the vagina is disordered, lactobacillus, particularly a strain producing hydrogen peroxide (H2O 2) is reduced, and other flora such as gardnerella vaginalis, curvulus mobilis, bacteroides, streptococcus digestus, mycoplasma hominis and the like are massively propagated to cause a comprehensive syndrome without inflammation of the vaginal mucosa. BV is cured for a long time and is easy to relapse, and the BV has direct relation with a plurality of serious obstetrics and gynecology complications, and can cause risk factors such as increased premature birth rate of BV patients, low infant physique, histic villitis, amniotic fluid infection, and endometritis. In addition, BV is also related to salpingitis, pelvic inflammatory disease, infertility, ectopic pregnancy, urinary infection, postoperative infection, gynecological tumor, etc. BV has been shown to increase the risk factor for HIV infection. Because of the high rate of incidence and the high recurrence of BV, many studies have shown that microbial and immunotherapy approaches, and the modulation of therapy by ecological agents is a necessary option for the treatment of such diseases. The probiotics is a living microorganism, so that the lactobacillus is orally taken or implanted in the vagina to restore the natural resistance to the invasion of foreign bacteria, promote the self-cleaning effect of the vagina and have good effect on curing and preventing the relapse of BV.
Although direct vaginal administration has good effect, the vaginal administration is not convenient enough, and many women have a use conflict psychology. The prior art has demonstrated that orally administered lactic acid bacteria partially enter the vagina, but with low efficiency.
Chinese patent 2012101725644 discloses a strain of lactobacillus casei (lactobacillus casei) LC-N235, which has been deposited at chinese type culture collection CCTCC at 2012, 5/10, with a deposit number: CCTCC M2012157. The known function of this strain is high lactic acid production. Lactobacillus casei (Lactobacillus casei) LC-N235 used in the invention is given by researchers of Nanjing university of industry.
The lactobacillus crispatus and the lactobacillus casei used in the invention are purchased from China general microbiological culture collection center. In the research and practice process of the invention group, the combination of oral lactobacillus crispatus CGMCC 1.2743 and lactobacillus casei (Lactobacillus casei) LC-N235 can effectively enhance the treatment effect on bacterial vaginitis, and remarkably restore vaginal microecological balance. Lactobacillus crispatus and Lactobacillus casei are safe probiotics and have a prospect as oral reagents. How the lactobacillus casei (lactobacillus casei) LC-N235 enhances the oral effect of lactobacillus crispatus CGMCC 1.2743 is not clear at present, and the synergy is supposed to be realized through a series of physiological and biochemical influences on the intestinal tract.
The technical problem to be solved by the invention can be realized by the following technical scheme.
A probiotic composition for use in modulating the balance of the gynecological flora comprising:
lactobacillus crispatus, lactobacillus casei LC-N235 and other acceptable pharmaceutical excipients.
The Lactobacillus crispatus is purchased and preserved in China general microbiological culture collection center with the preservation number of CGMCC 1.2743.
The lactobacillus casei (Lactobacillus casei) LC-N235 is preserved in China center for type culture Collection CCTCC (China center for type culture Collection) in 5-10.2012 with the preservation number: CCTCC M2012157.
The probiotic composition can be prepared into oral liquid, and can also be prepared into fermented food, preferably fermented milk, fermented soymilk and the like.
The invention has the advantages that:
the probiotic composition has definite and safe components, can be directly prepared into oral liquid or fermented food, is convenient to eat, has no conflict psychology for women, can be taken as health care for a long time, and has very important significance and wide prospect as a vaginal microecological balance appliance.
Detailed Description
The following examples of the present invention are described in detail, and are only for the purpose of illustrating the present invention and are not to be construed as limiting the present invention.
Specific examples of the present invention are described below.
Example 1
The mouse molding method comprises the following steps: 35 female mice weighing 20-22g were divided into 7 groups, and there was no statistical difference between the mice in each group. The 7 groups are respectively a blank control group (normal mice), a positive control group (levofloxacin group), a negative control group, a lactobacillus crispatus CGMCC 1.2743 treatment group, a lactobacillus casei LC-N235 treatment group, a lactobacillus crispatus LC-N235 treatment group and a lactobacillus casei crispatus treatment group. Each group had 5 mice. Wherein the normal mouse group is used as a blank control, and the positive control group, the negative control group and the treatment group are mice successfully modeled by bacterial vaginitis.
The molding method of the bacterial vaginitis comprises the following steps: the mice were given a daily vaginal injection of 0.3 mL of a mixed bacterial suspension prepared from Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa at a ratio of 1: 1 (the concentrations of the three bacteria are 0.5 x 10)7cfu/ml), after 7 days of continuous treatment, the vaginal orifice of the mouse is obviously red and swollen and pus flows out, and the modeling is successful.
The lactobacillus casei is purchased and preserved in China general microbiological culture collection center with the preservation number of CGMCC 18727.
After the modeling is successful, the blank control group and the negative control group are not administrated; the positive control group is administered with levofloxacin suspension for gastric lavage, and the dosage is converted into the dosage of mice according to the medication guidance, and the dosage contains levofloxacin solution 0.01 g/kg. The treatment groups activated lactobacillus, and three different viable bacteria suspensions were prepared, all with a concentration of 0.5 x 108cfu/ml, different groups were given corresponding bacteria for gastric lavage. Lactobacillus crispatus CGMCC 1.2743 treatment group, 500 mu L/d Lactobacillus crispatus for each mouse; lactobacillus casei LC-N235 treatment group: 500. mu.L/d Lactobacillus casei LC-N235 per mouse; curl + LC-N235 treatment group: 250. mu.L/d Lactobacillus crispatus + 250. mu.L/d Lactobacillus casei LC-N235 per mouse; crimp + lactobacillus casei treatment group: 250. mu.L/d Lactobacillus crispatus + 250. mu.L/d Lactobacillus casei per mouse; all groups were treated continuously for 7 days, and data on mice serum inflammatory factors and vaginal secretions were collected on day 8.
And (3) detecting inflammatory factors: after 7 days of treatment, blood was collected from orbital veins of mice on day 8, the blood was collected in an EP tube, left to stand at room temperature for 2 hours and centrifuged, and the upper serum was collected and tested for Procalcitonin (PCT), C-reactive protein (CRP), interleukin 6 (IL-6) and interleukin 10 (IL-10) in the serum of mice by the Elisa method described in the test kit. The test results are shown in table 1.
Statistical treatment: statistical analysis was performed using SPSS16.0 and the results of the data were expressed as mean ± standard deviation using the t test between groups.
TABLE 1 detection of inflammatory factors after treatment of different groups of mice
Note: t test, l: p <0.05 (compared to blank); 2: p <0.05 (compared to positive control); 3: p <0.05 (compare with negative control group)
After corresponding treatment, the detection of the mouse serum inflammatory factors of the positive control group, the negative control group and the treatment group is analyzed. The results show that: the mice in the group treated with lactobacillus casei LC-N235 had minimal Procalcitonin (PCT), C-reactive protein (CRP), interleukin 6 (IL-6), interleukin 10 (IL-10) levels, and had significant differences (< 0.05) relative to the negative control group, while the other treated groups had no significant differences relative to the negative control group; the lactobacillus treatment group alone has no obvious difference compared with the negative control group (P > 0.05); the results show that the treatment of the Lactobacillus casei and Lactobacillus casei LC-N235 has the function of synergistically eliminating in-vivo inflammatory factors, and the function is close to that of antibiotic treatment.
Example 2
After 7 days of treatment, the groups of example 1 were tested for vaginal flora after treatment on day 8:
detecting mouse vaginal flora by collecting 100ul vaginal rinse solution under aseptic condition, mixing with normal saline, sequentially diluting by 10 times, and selecting 10 times of original solution3、104、105、106Dilutions were plated on MRS, EMB, SP, LB media. After aerobic culture (Lactobacillus is subjected to anaerobic culture) for 48 hours at 37 ℃, counting colonies, calculating the viable count (cfu/ml) of the Lactobacillus, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa in each milliliter of vaginal secretion, and taking the number of the viable count for statistical treatment. The results are shown in Table 2.
TABLE 2 detection of vaginal flora after treatment of mice of different groups
Note: t test, l: p <0.05 (compared to blank); 2: p <0.05 (compared to positive control); 3: p <0.05 (compare with negative control group)
After corresponding treatment, the vaginal flora of the mice of the positive control group, the negative control group and the treatment group is treated
The changes were analyzed. The results show that: normal mice are also a state in which various bacteria coexist, and lactobacillus is relatively dominant. In the experiment, the mice in the curling + lactobacillus casei LC-N235 treatment group have the largest fixed planting amount of lactobacillus in the vagina, the fixed planting amounts of escherichia coli and staphylococcus aureus are the smallest, and the mice have significant differences (< 0.05) relative to a blank group, a positive control group and a negative control group, and are considered to be related to intestinal crawling fixed planting after short-time supplement of lactobacillus; the number of coliform bacteria and staphylococcus aureus in the lactobacillus casei and lactobacillus casei LC-N235 treatment group is statistically different (less than 0.05) relative to that in the negative control group, which indicates that the treatment of the lactobacillus has the effect of removing the coliform bacteria and the staphylococcus aureus from permanent planting in the vagina of the mouse; the positive control group has the least bacteria amount and takes the antibiotic sterilization and bacteriostasis into consideration; there was no significant difference in P.aeruginosa between groups (P > 0.05).
In conclusion, in the research and practice process of the team, the combination of oral lactobacillus crispatus and lactobacillus casei LC-N235 can effectively enhance the treatment effect on bacterial vaginitis and remarkably restore the vaginal microecological balance. Lactobacillus crispatus and Lactobacillus casei LC-N235 are safe probiotics and have a good prospect as oral reagents. The use of other Lactobacillus casei, such as Lactobacillus casei CGMCC 18727 listed in the examples, did not have this effect. It is not clear how the Lactobacillus casei LC-N235 enhances the oral effect of Lactobacillus crispatus, and it is presumed that the effect is synergistic through a series of physiological and biochemical effects on the intestinal tract.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (4)
1. A probiotic composition for use in modulating the balance of the gynecological flora comprising:
lactobacillus crispatus, lactobacillus casei (lactobacillus casei) LC-N235 and other acceptable pharmaceutical excipients;
the strain of lactobacillus crispatus is preserved in China center for type culture Collection with the preservation number: CGMCC 1.2743;
the lactobacillus casei LC-N235 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number: CCTCC M2012157.
2. The probiotic composition of claim 1 formulated as an oral liquid.
3. The probiotic composition of claim 1 formulated as a fermented food product.
4. The probiotic composition of claim 3 wherein said fermented food product is fermented milk, fermented soymilk.
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