Application of lactobacillus rhamnosus in preparation of product for treating delayed hypogonadism

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses application of lactobacillus rhamnosus fermented milk in a product for improving male functions. According to the application, the new application of the lactobacillus rhamnosus B6 (CGMCC No.13310) fermented milk in the aspect of improving male functions is disclosed for the first time, and the new application is specifically characterized by improving male fertility desire, reproductive capacity, seminiferous tubule dysfunction and spermatogenesis, testis oxidative stress activity, testis testosterone synthesis and sperm quality; the strain has a potential value of producing a gain substance for improving the male function by fermenting skimmed milk, and also prompts that the fermented milk has a potential application prospect for enhancing the male function.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to the application of Lacticaseibacillus rhamnosus in the preparation of a product for treating late-onset hypogonadism. BACKGROUND

[0002] Since the 21st century, the prevalence of infertility has been increasing year by year worldwide. About 7% of couples worldwide suffer from infertility, and the decline in male fertility at childbearing age has become a social problem that needs to be solved. A large number of studies have shown that after the age of 30, the bioavailable testosterone levels of men decrease linearly. With the decrease of testosterone levels, 13% of men further develop pathological symptoms in physical, sexual and cognitive functions, such as decreased bone and muscle mass, decreased sexual function, depression and cognitive impairment, etc., which we call late-onset hypogonadism (LOH).

[0003] LOH can have adverse effects on the function of multiple organs and the quality of life. The main intervention method at present is testosterone replacement therapy (TST), which can effectively alleviate the symptoms related to LOH and improve the quality of life, but TST is accompanied by a variety of side effects, including an increased susceptibility to stroke, heart attack and prostate tumors. Studies have found that the intestinal flora is closely related to male reproductive health, and the intestinal flora has the potential ability to affect testicular function, spermatogenesis and sex hormone metabolism through various ways.

[0004] Therefore, it is an urgent problem for those skilled in the art to explore a probiotic fermentation product that actively affects LOH and testosterone secretion and various male function indicators in male individuals under the condition of extremely low or no side effects through specific mechanisms. SUMMARY

[0005] In order to solve the above technical problems, the present application provides the application of Lacticaseibacillus rhamnosus in the preparation of a product for treating late-onset hypogonadism, and the preservation number of the Lacticaseibacillus rhamnosus is CGMCC NO.13310.

[0006] The strain preservation information of the present application is as follows:

[0007] Strain name: Lacticaseibacillus rhamnosus;

[0008] Preservation number: CGMCC No.13310;

[0009] Preservation date: November 15, 2016;

[0010] Depositary name: China General Microbiological Culture Collection Center;

[0011] Abbreviation of the depositary: CGMCC;

[0012] Address of the depositary: No. 1, Yihuan Road, Beijing, China.

[0013] The beneficial effects brought by the present application include but are not limited to: the new use of Lactobacillus rhamnosus B6 fermented milk in improving male function is disclosed for the first time, the potential value of the above-mentioned strain in producing gain substances for improving male function by fermenting skim milk is revealed, and the potential application prospect of the above-mentioned fermented milk in enhancing male function is also suggested. BRIEF DESCRIPTION OF DRAWINGS

[0014] The present application will be further illustrated in the form of exemplary embodiments, which will be described in detail with the aid of the accompanying drawings. These embodiments are not limiting, in which:

[0015] Figure 1 Effect of Lactobacillus rhamnosus fermented milk on male sexual desire (A) sniffing frequency, (B) sniffing time, (C) climbing latency, (D) climbing duration, (E) climbing frequency.

[0016] Figure 2 Effect of Lactobacillus rhamnosus fermented milk on male reproductive capacity (A) first litter latency, (B) average litter number, (C) average number of offspring per litter.

[0017] Figure 3 Effect of Lactobacillus rhamnosus fermented milk on male spermatogenic tubule dysfunction and spermatogenesis (A) seminiferous tubule diameter, (B) atrophic seminiferous tubule ratio, (C) Sycp3 transcription level, (D) Inha transcription level, (E) Nectin2 transcription level, (F) Creb1 transcription level.

[0018] Figure 4 Effect of Lactobacillus rhamnosus fermented milk on male testicular oxidative stress activity (A) SOD activity, (B) MDA content, (C) CAT activity, (D) Prx3 transcription level, (E) Prx4 transcription level, (F) Gstm5 transcription level, (G) Gpx4 transcription level.

[0019] Figure 5 Effect of Lactobacillus rhamnosus fermented milk on male testicular testosterone synthesis (A) serum GnRH level, (B) serum LH level, (C) serum TE level, (D) testicular TE level, (E) Star transcription level, (F) Hsd3b1 transcription level.

[0020] Figure 6Effect of Lactobacillus rhamnosus fermented milk on male sperm quality (A) sperm DIFF staining chart, (B) abnormal sperm ratio, (C) sperm number, (D) motile sperm ratio, (E) forward motile sperm ratio, (F) immotile sperm ratio, (G) rapid motile sperm ratio, (H) sperm average curvilinear velocity, (I) sperm average straight line velocity, (J) sperm average path velocity, (K) sperm head average lateral displacement amplitude, (L) sperm average beat cross frequency. DETAILED DESCRIPTION

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present specification, the drawings required to be used in the embodiment description will be briefly introduced as follows. Obviously, the drawings in the following description are only some examples or embodiments of the present specification, and for those skilled in the art, the present specification can also be applied to other similar scenarios without creative labor on the basis of these drawings. Unless the context clearly indicates otherwise or otherwise stated, the same reference numbers in the drawings represent the same structures or operations.

[0022] As shown in the specification and claims, unless the context clearly indicates otherwise or otherwise stated, the words "one", "a", "an", and / or "the" do not necessarily refer to the singular, but can also include the plural. Generally speaking, the terms "comprise" and "include" only indicate the inclusion of the steps and elements explicitly identified, and these steps and elements do not constitute an exclusive list, and the method or device can also include other steps or elements.

[0023] Flowcharts are used in the present specification to illustrate the operations performed by the system according to the embodiments of the present specification. It should be understood that the preceding or subsequent operations are not necessarily performed in sequence. On the contrary, each step can be processed in reverse order or simultaneously. At the same time, other operations can also be added to these processes, or one or more steps of operation can be removed from these processes.

[0024] The present application provides the use of Lactobacillus rhamnosus in the preparation of a product for treating delayed puberty, the Lactobacillus rhamnosus has a preservation number of CGMCC NO.13310.

[0025] In some embodiments, the product can contain Lactobacillus rhamnosus fermentation products.

[0026] In some embodiments, the product can improve male function.

[0027] In some embodiments, the product can improve male libido.

[0028] In some embodiments, the product can improve male reproductive capacity. In some embodiments, preferably, the product can improve the speed of female individuals getting pregnant, and improve the fertility potential of female individuals.

[0029] In some embodiments, the product can improve male seminiferous tubule dysfunction and spermatogenesis. In some embodiments, preferably, the product can regulate the diameter of seminiferous tubules and the number of atrophic seminiferous tubules of testis, and improve the transcription level of spermatogenesis-related genes Sycp3, Inha, Nectin2 or Creb1 of testis.

[0030] In some embodiments, the product can improve the oxidative stress activity of male testis. In some embodiments, preferably, the product can increase the activity of superoxide dismutase or catalase of testis, reduce the level of malondialdehyde of testis, and improve the transcription level of antioxidant genes Prx3, Prx4, Gstm5 or Gpx4 in testis.

[0031] In some embodiments, the product can improve the levels of gonadotropin-releasing hormone, luteinizing hormone in serum of male, and testosterone in serum and testis, and improve the expression of testosterone synthesis pathway genes Star and Hsd3b1 of testis.

[0032] In some embodiments, the product can improve the quality of sperm of male. In some embodiments, preferably, the product can improve any one or more of sperm count, percentage of motile sperm, percentage of progressively motile sperm, percentage of immotile sperm, percentage of rapidly motile sperm, average curvilinear velocity of sperm motility, average straight line velocity of sperm motility, average path velocity of sperm motility, average amplitude of lateral head displacement of sperm, or average flagellar beat frequency of sperm.

[0033] In some embodiments, the product comprises any one or more of food, medicine or health product.

[0034] In some embodiments, the dosage form of the product can comprise injection, suspension, powder, tablet or granule.

[0035] In some embodiments, the medicine can further comprise a pharmaceutically acceptable carrier.

[0036] In some embodiments, the effective amount of viable Lactobacillus rhamnosus in the product is not less than 1 x 10 9 CFU / kg / day.

[0037] In some embodiments, the product can be prepared by inoculating Lactobacillus rhamnosus in skimmed milk for fermentation.

[0038] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are all set up with three repeated experiments, and the results are averaged.

[0039] The Lacticaseibacillus rhamnosus strain B6 referred to in the following content, classified as Lacticaseibacillus rhamnosus, was deposited on November 15, 2016, with the accession number CGMCC No. 13310, and the depositing unit was the General Microbiological Center of China Microorganism Culture Collection Management Committee, located at No. 1, Beichen West Road, Haidian District, Beijing.

[0040] Preparation method of seed (fermentation strain):

[0041] The strain was picked up with an inoculation loop to put a single colony into 10 mL of MRS liquid medium (purchased from Merck Co., Germany), and a vortex shaker was used to uniformly disperse the colony in the liquid medium. After 48 h of anaerobic culture at 37°C, the culture was removed, inoculated into MRS liquid medium at an inoculation amount of 2% (v / v), and cultured anaerobically at 37°C for 24 h. Then, the culture was centrifuged at 15,000 rpm for 10 min, the supernatant was discarded, the bacterial cells were washed twice with sterile distilled water, and then suspended with sterile distilled water at the original culture volume to obtain seed for fermentation. The bacterial concentration of the seed liquid was 3.7 x 10 9 CFU / mL.

[0042] Preparation method of skim milk:

[0043] The skim milk powder with a mass percentage of 10% was mixed with distilled water, fully dissolved, sterilized at 125°C for 5 min, and cooled to room temperature to obtain the required concentration of skim milk.

[0044] Preparation method of fermented milk:

[0045] The fermentation seed liquid of Lacticaseibacillus rhamnosus CGMCC NO. 13310 was inoculated into skim milk (prepared in the material preparation with a mass percentage of 10%) at an inoculation amount of 5% (v / v, the same below), and cultured at 37°C for 20 h to obtain fermented milk (to ensure the uniformity of the gavage sample, the fermented milk was freeze-dried into powder and stored at -80°C).

[0046] Example 1 animal experiment

[0047] 1. LOH model determination: 40 SPF level 20-month-old male C57BL / 6 mice (Beijing Huabao) were purchased and acclimated for 1 week. On the last day of acclimation, serum total testosterone levels were measured in blood collected from the tail tip of each mouse using an ELISA kit between 8 am and 12 pm. C57BL / 6 mice with serum total testosterone levels below 10 ng / mL were considered to be a model of late-onset hypogonadism (LOH), while C57BL / 6 mice with serum total testosterone levels above 10 ng / mL were considered to be a non-LOH model.

[0048] 2. Grouping and intervention: 20 LOH model mice were randomly divided into 2 groups (one group was a model group, and the other group was a fermented milk group), one cage per mouse (n = 10). According to the testosterone level, the mice were randomly grouped using a random number table to ensure uniformity between groups. The specific intervention methods were as follows:

[0049] (1) Model group (Mod): 200 μl of saline was administered orally, and the administration was continued for 13 weeks.

[0050] (2) Fermented milk group (B6): Fermented milk freeze-dried powder was dissolved in 200 μl of saline, and the administration was continued at a dose of 1 x 10 9 CFU / kg / day for 13 weeks.

[0051] 3. Effect of L. rhamnosus-fermented milk on male sexual desire

[0052] At the beginning of the intervention experiment, a 6-week-old C57BL / 6 female mouse (Beijing Huabao) was placed in each cage, and the sexual desire indicators of each LOH model mouse were recorded and analyzed using an infrared camera within 30 minutes of introducing the female mouse: sniffing frequency, sniffing time, climbing latency (climbing the back of the female mouse), climbing duration, and climbing frequency.

[0053] The results, as shown in Figure 1 , showed that the sniffing frequency and sniffing time of LOH mice treated with L. rhamnosus-fermented milk showed an increasing trend Figure 1 (A-B), and the climbing latency of LOH mice in the intervention group was significantly shortened Figure 1 (C), the climbing duration was significantly prolonged Figure 1 (D), and the climbing frequency showed an increasing trend Figure 1 (E). In summary, L. rhamnosus-fermented milk significantly improved the sexual desire of male LOH mice.

[0054] 4. Effect of L. rhamnosus-fermented milk on male reproductive capacity

[0055] After the completion of the fertility experiment, 1 six-week-old C57BL / 6 female mouse was again placed in each cage, and the male mice were paired with 2 female mice for 9 weeks, and the first litter latency, total litter number and average number of offspring per litter of each group of mice were recorded.

[0056] The results are shown in Figure 2 A), and the total litter number and average number of offspring per litter showed a trend of increase Figure 2 A), and the total litter number and average number of offspring per litter showed a trend of increase Figure 2 B-C). In summary, the Lactobacillus rhamnosus fermented milk significantly improved the reproductive capacity of male LOH mice.

[0057] 5. Effect of Lactobacillus rhamnosus fermented milk on male spermatogenic tubule dysfunction and spermatogenesis

[0058] After the end of the experiment, isoflurane was used to anesthetize and sacrifice the mice in each group, and part of the testicular tissue was taken, the diameter of the spermatogenic tubule and the proportion of atrophic spermatogenic tubules were counted by PAS staining, and the transcription level of spermatogenesis-related genes (Sycp3, Inha, Nectin2 and Creb1) was detected by qRT-PCR method Top Green qPCRSuperMix(+Dye I)(Quanshijin:AQ132-11) kit).

[0059] The results are shown in Figure 3 PAS staining revealed that Lactobacillus rhamnosus fermented milk had a significant regulatory effect on the diameter of the spermatogenic tubule and the number of atrophic spermatogenic tubules in the testis of LOH model mice Figure 3 A-B). Further detection of the transcription level of spermatogenesis-related genes found that Lactobacillus rhamnosus fermented milk could significantly increase the transcription level of 4 spermatogenesis-related genes Sycp3, Inha, Nectin2 and Creb1 in the testis of LOH mice Figure 3 C-F). In summary, Lactobacillus rhamnosus fermented milk significantly improved the spermatogenic tubule dysfunction and spermatogenesis of male LOH mice.

[0060] 6. Effect of Lactobacillus rhamnosus fermented milk on the activity of oxidative stress in the testis of male mice

[0061] After the end of the experiment, isoflurane was used to anesthetize and sacrifice the mice in each group, and part of the testicular tissue was taken, the activity of superoxide dismutase (SOD) in the testis, the content of malondialdehyde (MDA) and the activity of catalase (CAT) were determined by ELISA kit, and the transcription level of testicular antioxidant-related genes (Prx3, Prx4, Gstm5 and Gpx4) was detected by qRT-PCR method.

[0062] The results are as follows Figure 4 As shown, LOH mice treated with Lactobacillus rhamnosus fermented milk exhibited significantly increased testicular SOD activity. Figure 4 A), while MDA levels decreased significantly ( Figure 4 B), and CAT activity increased significantly ( Figure 4 C). Transcriptional level analysis of antioxidant genes revealed that *Lactobacillus rhamnosus* fermented milk significantly increased the transcriptional levels of four antioxidant genes (Prx3, Prx4, Gstm5, and Gpx4) in the testes of LOH model mice. Figure 4 (DG). In summary, Lactobacillus rhamnosus fermented milk significantly improved testicular oxidative stress activation in male LOH mice.

[0063] 7. Effects of Lactobacillus rhamnosus fermented milk on testosterone synthesis in male testes

[0064] After the experiment, mice in each group were anesthetized and sacrificed using isoflurane. Serum and some testicular tissue were collected. The levels of gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), and testosterone (TE) in serum and testes were measured using an ELISA kit. The transcriptional level of key genes (Star and Hsd3b1) in the testicular testosterone synthesis pathway was detected using qRT-PCR.

[0065] The results are as follows Figure 5 As shown, serum GnRH, serum LH, and serum and testicular TE levels in LOH mice treated with Lactobacillus rhamnosus fermented milk all showed an increasing trend. Figure 5 Given that GnRH and LH are upstream regulators of testicular TE synthesis, and that they maintain physiological testosterone levels through a pulsatile secretion pattern along the HPG axis, it can be concluded that the increase in testicular TE levels induced by *Lactobacillus rhamnosus* fermented milk is achieved through regulation of the HPG axis. Furthermore, the increasing trend of Star, a key gene in the testicular TE synthesis pathway, and the significant increase in Hsd3b1 expression in LOH mice treated with *Lactobacillus rhamnosus* fermented milk were observed. Figure 5 The EF data corroborate the fact that testicular TE levels are increased. In conclusion, Lactobacillus rhamnosus fermented milk significantly improves testicular TE synthesis levels in male LOH mice, which is achieved through regulation of the HPG axis.

[0066] 8. Effects of Lactobacillus rhamnosus fermented milk on male sperm quality

[0067] After the end of the experiment, each group of mice was anesthetized and sacrificed using isoflurane, and the right testis of each mouse was taken and decapsulated. Two portions (50 mg each) were taken and placed in two tubes containing Medium 199 (Thermofisher, No. 11150059) and incubated at 37°C for 2 hours. Subsequently, the sperm were stained with DIFF and morphological analysis was performed, and the routine parameters and motility parameters of the semen were detected by Computer Aided Sperm Analysis (CASA).

[0068] The results are shown in Figure 6 Morphological analysis showed that the number of abnormal sperm in LOH mice intervened by R. rhamnosus fermented milk was significantly lower than that in model LOH mice Figure 6 CASA analysis initially showed that R. rhamnosus fermented milk intervention could significantly improve the characteristics and motility parameters of sperm in LOH mice, including sperm count, the proportion of motile sperm, the proportion of progressively motile sperm, the proportion of immotile sperm, the proportion of rapidly motile sperm, the average curve velocity of sperm motility, the average straight line velocity of sperm motility, the average path velocity of sperm motility, the average lateral displacement amplitude of sperm head, and the average flagellar beat frequency of sperm Figure 6 C-L). In summary, R. rhamnosus fermented milk significantly improved the sperm quality of male LOH mice.

[0069] The above detailed disclosure is merely exemplary and does not limit the present specification, as apparent to those skilled in the art, in view of the basic concepts described above. Although not explicitly stated herein, those skilled in the art can make various modifications, improvements and corrections to the present specification. Such modifications, improvements and corrections are suggested in the present specification, so they still belong to the spirit and scope of the exemplary embodiments of the present specification.

[0070] Meanwhile, specific words are used in the present specification to describe the embodiments of the present specification. As "one embodiment", "an embodiment", and / or "some embodiments" mean a certain feature, structure or characteristic related to at least one embodiment of the present specification. Therefore, it should be emphasized and noted that "an embodiment" or "one embodiment" or "one alternative embodiment" mentioned in different places in the present specification does not necessarily mean the same embodiment. In addition, certain features, structures or characteristics in one or more embodiments of the present specification can be properly combined.

[0071] In some embodiments, numbers that describe amounts, dimensions, and so forth, are used in the description of the embodiments. It should be understood that such numbers are used only to illustrate certain embodiments and that the scope of the embodiments is not limited to the numbers. In some examples, such numbers are modified by the modifier "about" or "approximately." Unless otherwise indicated, "about" or "approximately" means ±20% of the value stated. Accordingly, in some embodiments, numerical parameters in the specification and claims are approximations that can vary depending on the desired properties sought to be obtained by the individual embodiments. In some embodiments, numerical parameters are approximations that can vary from the numerical parameters set forth in the specification and claims. In some embodiments, numerical parameters should be considered in the context of the number of significant digits in which they are set forth. Although the numerical ranges and parameters setting forth the broad scope of the embodiments are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values set forth in the specific examples are provided to be as precise as practicable.

[0072] Finally, it should be understood that the embodiments described herein are merely exemplary of the principles of the embodiments. Other variations having essentially the same structure and function are within the scope of the embodiments. Accordingly, the embodiments described herein are not limited to that precisely as shown and described.

Claims

1. The application of *Lactobacillus rhamnosus* in the preparation of products for treating delayed-onset hypogonadism, wherein the accession number of *Lactobacillus rhamnosus* is CGMCC NO.13310.

2. The application as described in claim 1, characterized in that, The product contains fermentation products of Lactobacillus rhamnosus.

3. The application as described in claim 1, characterized in that, The product enhances male function.

4. The application as described in claim 1, characterized in that, The product improves male fertility.

5. The application as described in claim 1, characterized in that, The product improves male reproductive capacity; preferably, the product increases the rate of conception in female individuals and enhances their reproductive potential.

6. The application as described in claim 1, characterized in that, The product improves male seminiferous tubule dysfunction and spermatogenesis. Preferably, the product regulates the diameter of seminiferous tubules and the number of atrophied seminiferous tubules in the testes, and increases the transcription levels of spermatogenesis-related genes Sycp3, Inha, Nectin2, or Creb1 in the testes.

7. The application as described in claim 1, characterized in that, The product improves the oxidative stress activation of male testes. Preferably, the product increases the activity of superoxide dismutase or catalase in the testes, reduces the level of malondialdehyde in the testes, and increases the transcription level of antioxidant genes Prx3, Prx4, Gstm5, or Gpx4 in the testes.

8. The application as described in claim 1, characterized in that, The product increases the levels of gonadotropin-releasing hormone and luteinizing hormone in male serum, as well as testosterone levels in serum and testes, and increases the expression of genes Star and Hsd3b1 in the testicular testosterone synthesis pathway.

9. The application as described in claim 1, characterized in that, The product improves male sperm quality, preferably by improving sperm count, percentage of motile sperm, percentage of forward-moving sperm, percentage of stationary sperm, percentage of rapidly moving sperm, average curvilinear velocity of sperm, average linear velocity of sperm, average path velocity of sperm, average lateral displacement of sperm head, or average whiplash frequency of sperm.

10. The application as described in claim 1, characterized in that, The products include any one or more of the following: food, medicine, or health products.

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