Patent ID: 12251408

DETAILED DESCRIPTION

It should be noted that, the following detailed descriptions are all exemplary, and are intended to provide further descriptions of the present disclosure. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those usually understood by a person of ordinary skill in the art to which the present disclosure belongs.

It should be noted that the terms used herein are merely used for describing specific implementations, and are not intended to limit exemplary implementations of the present disclosure. As used herein, the singular form is intended to include the plural form, unless the context clearly indicates otherwise. In addition, it should further be understood that terms “comprise” and/or “include” used in this specification indicate that there are features, steps, operations, devices, components, and/or combinations thereof.

In a specific implementation of the present disclosure, the present disclosure provides a strain ofLactobacillus plantarumNice-02 preserved in CCTCC (China Center for Type Culture Collection) (address: Wuhan University, Luojiashan, Wuchang, Wuhan, Hubei Province) on Apr. 21, 2023 with a biological preservation number of CCTCC NO: M 2023571.

In a second aspect of the present disclosure, the present disclosure provides a microbial agent, at least including theL. plantarumand/or a fermentation metabolite of theL. plantarum.

Further, the microbial agent includes an inactivatedL. plantarumand/or a fermentation metabolite of the inactivatedL. plantarum. The fermentation metabolite includes but is not limited to exopolysaccharides, polypeptide and amino acid. In this case, the microbial agent is a postbiotic preparation.

Therefore, in a third aspect, the present disclosure provides a preparation method of the microbial agent. The preparation method includes:S1: activation culture: theLactobacillus plantarumNice-02 is subjected to plate streaking into an activation culture medium to obtain a pure strain.

The activation culture medium is prepared from 0.05 to 0.2% of casein, 0.05 to 0.2% of sialic acid, 0.3 to 1.0% of beef extracts, 0.5 to 2% of peptone, 0.05 to 0.2% of sodium acetate, 0.05 to 0.2% of potassium dihydrogen phosphate and 1.5 to 2.0% of agar powder, and a pH value is adjusted to 7.0.

More specifically, the activation culture medium is prepared from 0.1% of casein, 0.1% of sialic acid, 0.7% of beef extracts, 1% of peptone, 0.1% of sodium acetate, 0.1% of potassium dihydrogen phosphate and 2.0% of agar powder, and the pH value is adjusted to 7.0.

S2: primary seed solution preparation: a single colony of theL. plantarumNice-02 prepared in S1 is picked and placed into a first culture solution at 28 to 30° C. for stationary culture for 12 to 14 h to obtain a primary seed solution.the first culture solution is prepared from 0.05 to 0.2% of casein, 0.05 to 0.2% of sialic acid, 1 to 5% of fructooligosaccharide, 0.1 to 1% of yeast powder, 0.05 to 0.2% of sodium acetate and 0.05 to 0.2% of potassium dihydrogen phosphate, and a pH value is adjusted to 7.0.

More specifically, the first culture solution is prepared from 0.1% of casein, 0.1% of sialic acid, 3% of fructooligosaccharide, 0.5% of yeast powder, 0.1% of sodium acetate and 0.1% of potassium dihydrogen phosphate, and the pH value is adjusted to 7.0.

S3: secondary seed solution preparation: the primary seed solution is inoculated into a second culture solution according to an inoculation amount of 0.1 to 5% (preferably 0.5%, v/v) for stationary culture at 30° C. to 40° C. (preferably 37° C.) for 10 to 12 h to obtain a secondary seed solution.

The second culture solution is prepared from 0.1 to 1% of casein, 0.05 to 0.2% of sialic acid, 1 to 3% of fructooligosaccharide, 0.1 to 1% of yeast powder and 0.05 to 0.2% of Tween-80, and a pH value is natural.

More specifically, the second culture solution is prepared from 0.5% of casein, 0.1% of sialic acid, 1.5% of fructooligosaccharide, 0.5% of yeast powder and 0.1% of Tween-80, and the pH value is natural.

S4: tertiary seed solution preparation: the cultured secondary seed solution is inoculated into a seeding tank containing a third culture solution under an aseptic condition at an inoculation amount of 0.5 to 5% (preferably 2%), and stir culture is performed at a stirring speed of 20 to 50 r/min (preferably 30 r/min), a temperature of 30° C. to 40° C. (preferably 37° C.), a pH value of 7.0 and a tank pressure of 0.03 to 0.06 MPa (preferably 0.05 Mpa) to obtain a tertiary seed solution after the culture for 10 to 12 h.

The third culture solution is prepared from 0.5 to 5% of peptone, 1 to 5% of isomaltooligosaccharide, 0.1 to 0.5% of dipotassium phosphate, 0.5 to 5% of glycine, 0.1 to 1% of tyrosine, 0.05 to 0.2% of Tween-80 and 0.05 to 0.2% of a polyether defoamer, and a pH value is natural.

More specifically, the third culture solution is prepared from 1% of peptone, 4% of isomaltooligosaccharide, 0.2% of dipotassium phosphate, 1% of glycine, 0.5% of tyrosine, 0.1% of Tween-80 and 0.1% of a polyether defoamer, and the pH value is natural.

S5: L. plantarumprimary fermentation: the tertiary seed solution is inoculated into a fermentation tank containing a sterilized culture solution at an inoculation amount of 0.5 to 5% (preferably 2%), a temperature of 32° C., a stirring speed of 40 to 50 r/min (preferably 45 r/min) to be cultured for 5 to 6 h.

The culture solution is prepared from 3% of isomaltooligosaccharide, 0.2% of casein, 2% of peptone, 5% of fructooligosaccharide, 3% of water-soluble starch, 0.1% of Tween-80, 0.5% of glycine, 0.5% of tyrosine and 0.2% of a polyether defoamer, and a pH value is natural.

S6:L. plantarumsecondary fermentation: after 5 to 6 h, the fermentation temperature is lowered to 30° C., the stirring speed is accelerated to 50 to 70 r/min (preferably 60 r/min), the tank pressure is kept at 0.05 MPa, the pH value is kept natural, and fermentation is performed for 3 to 4 h.

S7:L. plantarumtertiary fermentation: after 8 to 10 h, the stirring speed is kept unchanged, the fermentation temperature is raised to 35° C., the tank pressure is kept at 0.05 MPa, the pH value is kept at 6.5, and fermentation is performed for 2 h.

S8:L. plantarumquaternary fermentation: after 10 to 12 h, the stirring speed is decelerated to 30 to 40 r/min (preferably 40 r/min), the fermentation temperature is raised to 40° C., the tank pressure is kept at 0.05 MPa, the pH value is kept at 6.5, and fermentation is performed for 1 to 2 h.

S9:L. plantarumquinary fermentation: after 11 to 14 h, the stirring speed is kept unchanged, the temperature is raised to 45° C., sterile lipoteichoic acid accounting for 0.05 to 0.2% (preferably 0.1%) of the volume of the fermentation solution is added into the fermentation tank, and the temperature is lowered to 20° C. for 30 min.

The preparation method further includes performing high-temperature inactivation and spray drying on the fermentation solution prepared in S9. The high-temperature inactivation specifically includes: inactivation treatment is performed after the state maintenance for 40 min at 75° C., and spray drying may be performed after temperature lowering. For the spray drying, an air inlet temperature is set to be 150° C. to 180° C., an air outlet temperature is set to be 90° C. to 100° C., and postbiotic preparation powder is prepared after the spray drying.

In another implementation of the present disclosure, the present disclosure provides application of the postbiotic preparation to preparation of a pelvic floor muscle rehabilitation product.

The pelvic floor muscle rehabilitation product may be food or medicine.

More specifically, the medicine may include at least one of other medicine inactive ingredients.

The medicine inactive ingredients may be carriers, excipients, diluents, etc. commonly used in medicine. Additionally, according to a general method, they may be made into an oral preparation of powder, granules, suspensions, emulsion, syrup, spray, etc.

The non-medical active ingredients such as the carriers, the excipients and the diluents capable of being included are well known in the field, and may be determined to conform to the clinical standard by those of ordinary skill in the art.

In another specific implementation of the present disclosure, the carriers, the excipients and the diluents include but are not limited to lactose, glucose, saccharose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, arabic gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, nipasol, talcum powder, magnesium stearate, mineral oil, etc.

The following embodiments are used to further explain the present disclosure, but are not intended to limit the present disclosure. It should be understood that the embodiments herein are provided for describing the present disclosure and not intended to limit the scope of the present disclosure. In the embodiments, “%” of ingredients in each culture medium (solution) represents mass percentage in volume, and the unit is g/mL.

Embodiment 1

A preparation method of postbiotic for pelvic floor muscle rehabilitation included:

On a clean bench,Lactobacillus plantarumNice-02 preserved in a −80° C. low-temperature refrigerator was subjected to plate streaking to a culture medium plate, a single colony was screened, and the single colony was repeatedly activated for 3 times to obtain a pure strain.

The composition of the culture medium was as follows: 0.1% of casein, 0.1% of sialic acid, 0.7% of beef extracts, 1% of peptone, 0.1% of sodium acetate, 0.1% of potassium dihydrogen phosphate and 2.0% of agar powder were contained in per 100 mL of the culture medium, and a pH value was adjusted to 7.0 with potassium hydroxide.

The single colony was picked and placed into a 100 mL of a liquid culture medium (the culture medium was placed into a 15*200 test tube. The composition of the liquid culture medium was as follows: 0.1% of casein, 0.1% of sialic acid, 3% of fructooligosaccharide, 0.5% of yeast powder, 0.1% of sodium acetate and 0.1% of potassium dihydrogen phosphate were contained in per 100 mL of the culture medium, and a pH value was adjusted to 7.0 with potassium hydroxide), and stationary culture was performed at 30° C. for 12 h.

The above cultured seed solution was inoculated into a triangular flask culture medium at an inoculation amount of 0.5%. That was, 0.5 mL of the primary seed solution was inoculated into per 100 mL of the secondary seed culture solution (0.5% of casein, 0.1% of sialic acid, 1.5% of fructooligosaccharide, 0.5% of yeast powder and 0.1% of Tween-80 were contained in per 100 mL of the culture solution). After the inoculation, a bottle opening was sealed by sterile gauze and kraft paper, the inoculated strain was uniformly mixed with the culture solution through slight shaking by hand. Then, the mixture was placed into a thermostat for stationary culture under the condition of 37° C. for 12 h. The obtained solution was called as the secondary seed solution.

The cultured secondary seed solution was inoculated into a seeding tank of a tertiary seed culture solution (1% of peptone, 4% of isomaltooligosaccharide, 0.2% of dipotassium phosphate, 1% of glycine, 0.5% of tyrosine, 0.1% of Tween-80 and 0.1% of a polyether defoamer were contained in per 100 mL of the culture medium, and a pH value was natural) under the aseptic condition at an inoculation amount of 2% (v/v), and stir culture was performed at a stirring speed of 30 r/min, a temperature of 37° C., a pH value of 7.0 and a tank pressure of 0.05 MPa for 12 h.

The tertiary seed solution was inoculated into a fermentation tank containing a sterilized fermentation solution (3% of isomaltooligosaccharide, 0.2% of casein, 2% of peptone, 5% of fructooligosaccharide, 3% of water-soluble starch, 0.1% of Tween-80, 0.5% of glycine, 0.5% of tyrosine and 0.2% of a polyether defoamer were contained in per 100 mL of the culture medium, and a pH value was natural) at an inoculation amount of 2% (v/v), a temperature of 32° C. and a stirring speed of 45 r/min to be cultured for 5 h. After 5 h, the fermentation temperature was lowered to 30° C., the stirring speed was raised to 60 r/min, the tank pressure was kept at 0.05 MPa, the pH value was natural, and the fermentation was performed for 3 h. After 8 h, the stirring speed was unchanged, the fermentation temperature was raised to 35° C., the tank pressure was kept at 0.05 MPa, the pH value was kept at 6.5 through an automatic pH regulation and control system, and the fermentation was performed for 2 h. After 10 h, the stirring speed was lowered to 40 r/min, the fermentation temperature was raised to 40° C., the tank pressure was kept at 0.05 MPa, the pH value was kept at 6.5 through an automatic pH regulation and control system, and the fermentation was performed for 1h. After 11 h, the stirring speed was unchanged, the temperature was raised to 45° C., sterile lipoteichoic acid accounting for 0.1% of the fermentation tank was added into the fermentation tank, the temperature was lowered to 20° C. for 30 min, then, the temperature was raised to 75° C. for 40 min for inactivation, and next, the temperature was lowered to 30° C.

An air inlet temperature was set to be 150° C. to 180° C., an air outlet temperature was set to be 90° C. to 100° C., and postbiotic powder was obtained after spray drying.

The postbiotic obtained according to Embodiment 1 contained 24% of exopolysaccharides, 20.8% of polypeptide and 38.5% of amino acid.

Embodiment 2

A preparation method of postbiotic for pelvic floor muscle rehabilitation included:

On a clean bench,Lactobacillus plantarumNice-02 preserved in a −80° C. low-temperature refrigerator was subjected to plate streaking to a culture medium plate, a single colony was screened, and the single colony was repeatedly activated for 3 times to obtain a pure strain.

The composition of the culture medium was as follows: 0.1% of casein, 0.1% of sialic acid, 0.7% of beef extracts, 1% of peptone, 0.1% of sodium acetate, 0.1% of potassium dihydrogen phosphate and 2.0% of agar powder were contained in per 100 mL of the culture medium, and a pH value was adjusted to 7.0 with potassium hydroxide.

The single colony was picked and placed into a 100 mL of a liquid culture medium (the culture medium was placed into a 15*200 test tube. The composition of the liquid culture medium was as follows: 0.1% of casein, 0.1% of sialic acid, 3% of fructooligosaccharide, 0.5% of yeast powder, 0.1% of sodium acetate and 0.1% of potassium dihydrogen phosphate were contained in per 100 mL of the culture medium, and a pH value was adjusted to 7.0 with potassium hydroxide), and stationary culture was performed at 30° C. for 12 h.

The above cultured seed solution was inoculated into a triangular flask culture medium at an inoculation amount of 0.5%. That was, 0.5 mL of the primary seed solution was inoculated into per 100 mL of the secondary seed culture solution (0.5% of casein, 0.1% of sialic acid, 1.5% of fructooligosaccharide, 0.5% of yeast powder and 0.1% of Tween-80 were contained in per 100 mL of the culture solution). After the inoculation, a bottle opening was sealed by sterile gauze and kraft paper, the inoculated strain was uniformly mixed with the culture solution through slight shaking by hand. Then, the mixture was placed into a thermostat for stationary culture under the condition of 37° C. for 12 h. The obtained solution was called as the secondary seed solution.

The cultured secondary seed solution was inoculated into a seeding tank of a tertiary seed culture solution (1% of peptone, 4% of isomaltooligosaccharide, 0.2% of dipotassium phosphate, 1% of glycine, 0.5% of tyrosine, 0.1% of Tween-80 and 0.1% of a polyether defoamer were contained in per 100 mL of the culture medium, and a pH value was natural) under the aseptic condition at an inoculation amount of 2%, and stir culture was performed at a stirring speed of 30 r/min, a temperature of 37° C., a pH value of 7.0 and a tank pressure of 0.05 MPa for 12 h.

The tertiary seed solution was inoculated into a fermentation tank containing a sterilized fermentation solution (3% of isomaltooligosaccharide, 0.2% of casein, 2% of peptone, 5% of fructooligosaccharide, 3% of water-soluble starch, 0.1% of Tween-80, 0.5% of glycine, 0.5% of tyrosine and 0.2% of a polyether defoamer were contained in per 100 mL of the culture medium, and a pH value was natural) at an inoculation amount of 2%, a temperature of 32° C. and a stirring speed of 45 r/min to be cultured for 46 h. After 46 h, the stirring speed was unchanged, the temperature was raised to 45° C., sterile lipoteichoic acid accounting for 0.1% of the fermentation tank was added into the fermentation tank, the temperature was lowered to 20° C. for 30 min, then, the temperature was raised to 75° C. for 40 min for inactivation, and next, the temperature was lowered to 30° C.

An air inlet temperature was set to be 150° C. to 180° C., an air outlet temperature was set to be 90° C. to 100° C., and postbiotic powder was obtained after spray drying.

The postbiotic obtained according to Embodiment 2 contained 19% of exopolysaccharides, 17.8% of polypeptide and 34% of amino acid.

Effect Verification

1. In Vitro Muscle Contraction Evaluation

Duodenum smooth muscles were used as experiment objects, and the influence of the postbiotic products on the duodenum smooth muscle contraction performance was observed and determined. Experiment scheme: rats were divided into a control group and experiment groups. The rats in the control group were normally fed, the rats in experiment groups were additionally fed with 0.5% (w w), 1.0% and 1.5% postbiotic (all products prepared according to Embodiment 1, and the three experiment groups were sequentially named as Experiment groups 1 to 3) in food. After the feeding for about 2 weeks, the rats were narcotized and killed through neck dislocation. The rat's abdomens were cut open to take intestinal canals by using stomachus pyloricus and duodenum junctions as starting points, the intestinal canals were cut into intestinal segments about 1 cm, were cleanly washed by a Krebs solution, and were placed into a Krebs solution, the moisture was preserved at a constant temperature of 37° C., the basic tension was adjusted to 1 g, and mixed gas (950 mL L−1oxygen and 50 mL·L−1carbon dioxide) was continuously introduced by using an air needle at a speed of 1 to 2 air bubbles per second. Duodenum smooth muscle contraction motion and change signals were introduced to a BL-420 Biological function test multi-channel physiological signal collection and processing system through a tonotransducer, and contraction curves were observed. The results were as shown in Table 1 below. The results in Table 1 show that the postbiotic has the function of promoting smooth muscle contraction, and shows certain dose dependency.

TABLE 1Influence of postbiotic on rat isolated intestinal smooth musclecontraction (x± s, n = 8)GroupAmplitude (g)Frequency (hz)Control group0.81 ± 0.140.57 ± 0.11Experimental group 11.61 ± 0.261.04 ± 0.23Experimental group 21.95 ± 0.321.412 ± 0.29Experimental group 31.72 ± 0.311.24 ± 0.41
2. Small-Scale Clinical Experiments on Pelvic Floor Muscle Injury Therapy

20 females with the ages between 40 to 60 participated in experiments on their own will, and completed questionnaire forms and records. People seriously troubled by stress urinary incontinence caused by pelvic floor muscle problems were in a test group. They orally took 600 mg of postbiotic (the product prepared according to Embodiment 1) on an empty stomach every day in the morning for 3 weeks. In the period, they did rope skipping to test the urine leakage condition. The original dietary habits were not changed in the test period, and the diet was normal.

After the use cycle was completed, 100% of postbiotic users expressed that they hardly adopted the jumping exercise mode before postbiotic use because urine leakage might occur after a few times of jumping, but after the postbiotic use, no urine leakage occurred in the whole rope skipping process, and they even had no urine leakage feeling.

TABLE 2Experiment statistical table of pelvic floormuscle rehabilitation people by postbioticSerialConditionnumberbefore takingDosage and use cycleCondition after taking1Urine leakage600 mg/day for 2 weeksNo urine leakage after 1500during jumpingtimes of rope skipping2Urine leakage600 mg/day for 2 weeksNo urine leakage after 1500during jumpingtimes of rope skipping3Urine leakage600 mg/day for 2 weeksNo urine leakage after 1500during jumpingtimes of rope skipping4Urine leakage600 mg/day for 2 weeksNo urine leakage after 1500during jumpingtimes of rope skipping5Urine leakage600 mg/day for 2 weeksSlight urine leakage after 3during joggingkm of jogging6Urine leakage600 mg/day for 2 weeksNo urine leakage duringduring coughsevere cough7Urine leakage600 mg/day for 2 weeksSlight urine leakage after 3during joggingkm of jogging8Urine leakage600 mg/day for 2 weeksSlight urine leakage afterduring jumping1500 times of rope skipping9Urine leakage600 mg/day for 2 weeksNo urine leakage duringduring coughsevere cough10Urine leakage600 mg/day for 2 weeksNo urine leakage duringduring coughsevere cough11Urine leakage600 mg/day for 2 weeksNo urine leakage after 3 kmduring joggingof jogging12Urine leakage600 mg/day for 2 weeksNo urine leakage after 3 kmduring joggingof jogging13Urine leakage600 mg/day for 2 weeksNo urine leakage after 1500during jumpingtimes of rope skipping14Urine leakage600 mg/day for 2 weeksNo urine leakage after 1500during jumpingtimes of rope skipping15Urine leakage600 mg/day for 2 weeksSlight urine leakage duringduring coughsevere cough16Urine leakage600 mg/day for 2 weeksNo urine leakage duringduring coughsevere cough17Urine leakage600 mg/day for 2 weeksNo urine leakage after 3 kmduring joggingof jogging18Urine leakage600 mg/day for 2 weeksNo urine leakage after 3 kmduring joggingof jogging19Urine leakage600 mg/day for 2 weeksNo urine leakage after 1500during jumpingtimes of rope skipping20Urine leakage600 mg/day for 2 weeksNo urine leakage after 1500during jumpingtimes of rope skipping

Through the above effect verification experiments, it is showed that the postbiotic preparation prepared by the present disclosure has a strong pelvic floor muscle rehabilitation function.

Finally, it should be finally noted that the foregoing descriptions are merely exemplary embodiments of the present disclosure, but are not intended to limit the present disclosure. Although the present disclosure has been described in detail with reference to the foregoing embodiments, for a person of ordinary skill in the art, modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent replacements can be made to some technical features in the technical solutions. Any modification, equivalent replacement, or improvement made and the like within the spirit and principle of the present disclosure shall fall within the protection scope of the present disclosure. The specific implementations of the present disclosure are described above, but are not intended to limit the protection scope of the present disclosure. A person skilled in the art should understand that various modifications or deformations may be made without creative efforts based on the technical solutions of the present disclosure, and such modifications or deformations shall fall within the protection scope of the present disclosure.