Compound microbial agent and application thereof

Abstract

The invention relates to a compound microbial agent and application thereof, belongs to the technical field of microorganisms, and particularly discloses application of mixed fermented milk in a product for improving male functions. According to the application, the new application of the mixed fermented milk of the rhamnosus lactobacillus B6 and the streptococcus thermophilus XJ53 (CGMCC No.17323) 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] This application relates to the field of biotechnology, and in particular to a compound microbial agent and its application. Background Technology

[0002] Since the beginning of the 21st century, the prevalence of infertility has been rising globally. Worldwide, approximately 7% of couples suffer from infertility, and declining fertility among men of reproductive age has become a pressing social problem. Numerous studies have shown that after age 30, men's bioavailable testosterone levels decline linearly. With this decline in testosterone levels, 13% of men experience further pathological symptoms related to physical, sexual, and cognitive functions, such as decreased bone and muscle mass, reduced sexual function, depression, and cognitive impairment; this condition is termed late-onset hypogonadism (LOH).

[0003] LOH (low-intensity toxicity) can adversely affect the function of multiple organs and quality of life. The main intervention currently available is testosterone replacement therapy (TST), which can effectively alleviate LOH-related symptoms and improve quality of life. However, TST is accompanied by various side effects, including increased susceptibility to stroke, heart attack, and prostate cancer. Research has found a close relationship between gut microbiota and male reproductive health, with the potential to influence testicular function, spermatogenesis, and sex hormone metabolism through multiple mechanisms.

[0004] Therefore, exploring a probiotic fermentation product that, through a specific mechanism, positively influences LOH and testosterone secretion and various male function indicators in male individuals under conditions of extremely low or no side effects is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] To address the aforementioned technical problems, this application provides a composite microbial inoculant comprising Lactobacillus rhamnosus and Streptococcus thermophilus, wherein the Lactobacillus rhamnosus has the preservation number CGMCC No. 13310 and the Streptococcus thermophilus has the preservation number CGMCC No. 17323.

[0006] This application also provides a fermented milk, which is produced by fermentation using the above-mentioned compound microbial agent.

[0007] This application also provides the use of the above-mentioned compound microbial agents or fermented milk in the preparation of products for treating delayed-onset hypogonadism.

[0008] The beneficial effects of this application include, but are not limited to: the first disclosure of a novel use of mixed fermented milk of Lactobacillus rhamnosus B6 and Streptococcus thermophilus XJ53 in improving male function, revealing the potential value of the above strains in producing beneficial substances that improve male function through fermentation of skim milk, and also suggesting the potential application prospects of the above fermented milk in enhancing male function. Attached Figure Description

[0009] This application will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein:

[0010] Figure 1 The effects of mixed fermented milk on male fertility desire: (A) sniffing frequency, (B) sniffing time, (C) climbing latency, (D) climbing duration, (E) climbing frequency.

[0011] Figure 2 The effects of mixed fermented milk on male reproductive capacity: (A) latency period of the first litter, (B) average number of litters, (C) average number of pups per litter.

[0012] Figure 3 Effects of mixed fermented milk on male seminiferous tubule dysfunction and spermatogenesis: (A) seminiferous tubule diameter, (B) percentage of atrophic seminiferous tubules, (C) Sycp3 transcription level, (D) Inha transcription level, (E) Nectin2 transcription level, (F) Creb1 transcription level.

[0013] Figure 4 Effects of mixed fermented milk on oxidative stress activation in male testes: (A) SOD activity, (B) MDA content, (C) CAT content, (D) Prx3 transcription level, (E) Prx4 transcription level, (F) Gstm5 transcription level, (G) Gpx4 transcription level.

[0014] Figure 5 Effects of mixed fermented milk on testosterone synthesis in male testes: (A) serum GnRH level, (B) serum LH level, (C) serum TE level, (D) testosterone TE level, (E) Star transcription level, (F) Hsd3b1 transcription level, (G) Hsd17b1 transcription level, (H) P450scc transcription level.

[0015] Figure 6Effects of mixed fermented milk on male sperm quality: (A) Sperm DIFF staining pattern, (B) Percentage of abnormal sperm, (C) Sperm count, (D) Percentage of motile sperm, (E) Percentage of forward-moving sperm, (F) Percentage of stationary sperm, (G) Percentage of rapidly moving sperm, (H) Average curvilinear velocity of sperm, (I) Average linear velocity of sperm, (J) Average path velocity of sperm, (K) Average lateral displacement amplitude of sperm head, (L) Average whiplash frequency of sperm. Detailed Implementation

[0016] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort. Unless obvious from the context or otherwise specified, the same reference numerals in the drawings represent the same structures or operations.

[0017] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not specifically refer to the singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0018] Flowcharts are used in this specification to illustrate the operations performed by the system according to embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed in exact order. Instead, the steps can be processed in reverse order or simultaneously. Furthermore, other operations can be added to these processes, or one or more steps can be removed from them.

[0019] This application provides a compound microbial inoculant, comprising Lactobacillus rhamnosus and Streptococcus thermophilus, wherein the Lactobacillus rhamnosus has the accession number CGMCC No. 13310 and the Streptococcus thermophilus has the accession number CGMCC No. 17323.

[0020] In some embodiments, the effective viable count ratio of *Lactobacillus rhamnosus* to *Streptococcus thermophilus* can be (80-95):(0.5-1). In some embodiments, the effective viable count ratio of *Lactobacillus rhamnosus* to *Streptococcus thermophilus* can be (82-93):(0.55-0.85). In some embodiments, the effective viable count ratio of *Lactobacillus rhamnosus* to *Streptococcus thermophilus* can be (84-91):(0.6-0.8). In some embodiments, the effective viable count ratio of *Lactobacillus rhamnosus* to *Streptococcus thermophilus* can be (86-99):(0.65-0.75). In some embodiments, the effective viable count ratio of *Lactobacillus rhamnosus* to *Streptococcus thermophilus* can be (88-99):(0.7-0.75).

[0021] In some embodiments, preferably, the effective viable count ratio of Lactobacillus rhamnosus to Streptococcus thermophilus can be 92.5:0.8.

[0022] This application also provides a fermented milk, which is produced by fermentation using the above-mentioned compound microbial agent.

[0023] In some embodiments, the fermented milk may further include an arginine solution. In some embodiments, preferably, the arginine concentration in the fermented milk is 0.1% to 0.5% g / mL. In some embodiments, preferably, the arginine concentration in the fermented milk is 0.15% to 0.45% g / mL. In some embodiments, preferably, the arginine concentration in the fermented milk is 0.20% to 0.40% g / mL. In some embodiments, preferably, the arginine concentration in the fermented milk is 0.25% to 0.35% g / mL. In some embodiments, preferably, the arginine concentration in the fermented milk is 0.30% to 0.35% g / mL.

[0024] In some embodiments, more preferably, the concentration of arginine in the fermented milk may be 0.25% g / mL.

[0025] In some embodiments, the arginine solution may be an aqueous solution of arginine.

[0026] This application also provides the use of the above-mentioned compound microbial agents or fermented milk in the preparation of products for treating delayed-onset hypogonadism.

[0027] In some embodiments, the product can enhance male function.

[0028] In some embodiments, the product can improve male fertility.

[0029] In some embodiments, the product can improve male fertility. In some embodiments, preferably, the product can increase the rate of conception in females and enhance their reproductive potential.

[0030] In some embodiments, the product can improve male seminiferous tubule dysfunction and spermatogenesis. In some embodiments, preferably, the product can regulate the diameter and number of atrophied seminiferous tubules in the testes, and increase the transcription levels of spermatogenesis-related genes Sycp3, Inha, Nectin2, or Creb1 in the testes.

[0031] In some embodiments, the product can improve oxidative stress activation in male testes. Preferably, in some embodiments, the product can increase the activity of testicular superoxide dismutase or catalase, decrease testicular malondialdehyde levels, and increase the transcriptional levels of antioxidant genes Prx3, Prx4, Gstm5, or Gpx4 in the testes.

[0032] In some embodiments, the product can increase the levels of gonadotropin-releasing hormone, luteinizing hormone, and testosterone in male serum and testes, and increase the expression of genes Star, Hsd3b1, Hsd17b1, and P450scc in the testicular testosterone synthesis pathway.

[0033] In some embodiments, the product can improve male sperm quality. In some embodiments, preferably, the product can improve any one or more of the following: 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 motility, average linear velocity of sperm motility, average path velocity of sperm motility, average lateral displacement of sperm head, or average whiplash frequency of sperm.

[0034] In some embodiments, the product may include one or more of food, medicine, or health products.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0036] The following information pertains to *Lactobacillus rhamnosus* strain B6, classified as *Lactobacillus rhamnosus*, deposited on November 15, 2016, with accession number CGMCC No. 13310, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The following information also pertains to *Streptococcus thermophilus* strain XJ53, classified as *Streptococcus thermophilus*, deposited on March 8, 2019, with accession number CGMCC No. 17323, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0037] Method for preparing seeds (fermentation starter):

[0038] A single colony of *Lactobacillus rhamnosus* CGMCC No. 13310 was picked up using an inoculation loop and placed in 10 mL of MRS liquid medium (purchased from Merck Co., Germany). The colony was evenly dispersed in the liquid medium using a vortex mixer and anaerobically cultured at 37°C for 48 h. The colony was then removed and inoculated into MRS liquid medium at a 2% (v / v) inoculation rate. After anaerobic culture at 37°C for 24 h, the culture was centrifuged at 15,000 rpm for 10 min, the supernatant was discarded, and the cells were washed twice with sterile distilled water. The cells were then resuspended in the original culture volume of sterile distilled water to obtain the seed culture for fermentation. The concentration of the seed culture was 3.7 × 10⁻⁶. 9 CFU / mL.

[0039] The lyophilized powder of *Streptococcus thermophilus* CGMCC No. 17323 was dissolved in a small amount of sterile distilled water. A loopful was used to streak a single colony onto M17 solid medium. After anaerobic incubation at 37°C for 48 hours, the colony was removed and picked up with an inoculation loop. A single colony was then placed into 10 mL of M17 liquid medium, and the colony was evenly dispersed using a vortex mixer. After anaerobic incubation at 37°C for 48 hours, the colony was inoculated into M17 liquid medium at a 2% (v / v) inoculation rate. After anaerobic incubation at 37°C for 24 hours, the culture was centrifuged at 15,000 rpm for 10 minutes. The supernatant was discarded, and the bacterial cells were washed twice with sterile distilled water and then resuspended in the original culture volume of sterile distilled water to obtain the fermentation seed culture. The concentration of *Streptococcus thermophilus* CGMCC No. 17323 in this seed culture was 8 x 10⁻⁶. 8 CFU / mL.

[0040] Preparation method of skim milk:

[0041] Mix 10% (by weight) of skim milk powder with distilled water until fully dissolved, then sterilize at 125°C for 5 minutes and cool to room temperature to obtain skim milk of the desired concentration.

[0042] Prepare a 10% arginine solution, filter it through a filter membrane to sterilize it, and set it aside for later use.

[0043] Methods for preparing fermented milk:

[0044] The fermentation seed culture of *Lactobacillus rhamnosus* CGMCC NO.13310 was aseptically inoculated into skim milk (prepared as a 10% mass percentage in the material preparation) at an inoculation amount of 2.5% (v / v, the volume percentage of seed culture to fermentation broth, the same below) and the fermentation seed culture of *Streptococcus thermophilus* CGMCC No.17323 at an inoculation amount of 0.1%. Then, sterile arginine solution (the final concentration of arginine in the fermented milk was 0.25%) was added at 10% of the fermentation volume, mixed well, and incubated at 37°C for 20 hours to obtain fermented milk (to ensure the homogeneity of the gavage sample, the fermented milk was freeze-dried into powder and frozen at -80°C for later use).

[0045] Example 1: Animal Experiment

[0046] 1. Determination of LOH Model: Forty SPF-grade 20-month-old male C57BL / 6 mice (from Shulaibao Company) were purchased and acclimatized for one week. On the last day of acclimatization, between 8:00 AM and 12:00 PM, serum total testosterone levels were measured using an ELISA kit on blood collected from the tail tip of the mice. C57BL / 6 mice with serum total testosterone levels below 10 ng / mL were considered to have a late-onset hypogonadism (LOH) model, while C57BL / 6 mice with serum total testosterone levels above 10 ng / mL were considered to have a non-LOH model.

[0047] 2. Grouping and Intervention: Twenty LOH model mice were randomly divided into two groups (one model group and the other fermented milk group), with one mouse per cage (n=10). Grouping was based on testosterone levels using a random number table to ensure homogeneity between groups. Specific intervention methods are as follows:

[0048] (1) Model group (Mod): 200 μl of physiological saline was administered by gavage for 13 weeks.

[0049] (2) Fermented milk group (B6XJA): The lyophilized fermented milk powder was dissolved in 200 μl of physiological saline, and then diluted with 1×10⁻⁶ mol / L. 9 CFU / kg / day dose administered via gavage for 13 weeks.

[0050] 3. The effect of mixed fermented milk on male fertility

[0051] In the fifth week of the intervention trial, a 6-week-old female C57BL / 6 mouse (from Shulaibao Company) was placed in each cage. Infrared cameras were used to record and analyze various fertility indicators of the LOH model mice in each group within 30 minutes after the introduction of the female mouse: sniffing frequency, sniffing time, climbing latency (climbing onto the back of the female mouse), climbing duration and climbing frequency.

[0052] The results are as follows Figure 1 As shown, the frequency and duration of sniffing of female mice by LOH mice treated with mixed fermented milk were significantly increased. Figure 1 AB), while the climbing latency of LOH mice in the intervention group was significantly shortened ( Figure 1 C), the duration of climbing was significantly prolonged ( Figure 1 D), climbing frequency increased significantly ( Figure 1 E). In conclusion, mixed fermented milk significantly improved fertility in male LOH mice.

[0053] 4. The effect of mixed fermented milk on male reproductive capacity

[0054] After the fertility experiment was completed, one 6-week-old female C57BL / 6 mouse was placed in each cage again, and the male mice were paired with two female mice for 9 weeks. The latency period of the first litter, the total number of litters, and the number of pups per litter were recorded for each group of mice.

[0055] The results are as follows Figure 2 As shown, the LOH model mice treated with mixed fermented milk had a shorter latency period in their first litter. Figure 2 A) The total number of litters and the average number of pups per litter were higher ( Figure 2 (BC). In conclusion, mixed fermented milk significantly improved the reproductive capacity of male LOH mice.

[0056] 5. Effects of mixed fermented milk on male seminiferous tubule dysfunction and spermatogenesis.

[0057] After the experiment, mice in each group were anesthetized and sacrificed using isoflurane. Testicular tissue was collected, and the diameter of seminiferous tubules and the percentage of atrophied seminiferous tubules were determined using PAS staining. The results were then analyzed using qRT-PCR. The Top Green qPCR SuperMix (+Dye I) (Quan Shi Jin: AQ132-11) kit was used to detect the transcriptional levels of spermatogenesis-related genes (Sycp3, Inha, Nectin2, and Creb1).

[0058] The results are as follows Figure 3 As shown, PAS staining revealed the significant regulatory effect of mixed fermented milk on the seminiferous tubule diameter and the number of atrophied seminiferous tubules in the testes of LOH model mice. Figure 3AB). Further analysis of the transcriptional levels of spermatogenesis-related genes revealed that mixed fermented milk significantly increased the transcriptional levels of four spermatogenesis-related genes—Sycp3, Inha, Nectin2, and Creb1—in the testes of LOH mice. Figure 3 (CF). In conclusion, mixed fermented milk significantly improved seminiferous tubule dysfunction and spermatogenesis in male LOH mice.

[0059] 6. Effects of mixed fermented milk on oxidative stress activation in male testes

[0060] After the experiment, mice in each group were anesthetized and sacrificed using isoflurane. Testicular tissue was collected, and the activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA), a marker of oxidative stress response, and the activity of catalase (CAT) in the testes were measured using an ELISA kit. The transcriptional level of antioxidant-related genes (Prx3, Prx4, Gstm5, and Gpx4) in the testes was detected by qRT-PCR.

[0061] The results are as follows Figure 4 As shown, the SOD activity in the testes of LOH mice treated with mixed fermented milk was significantly increased. 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 mixed 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, mixed fermented milk significantly improved testicular oxidative stress activation in male LOH mice.

[0062] 7. Effects of mixed fermented milk on testosterone synthesis in male testes

[0063] 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 levels of key genes (Star, Hsd3b1, Hsd17b1, P450scc) in the testicular testosterone synthesis pathway were detected using qRT-PCR.

[0064] The results are as follows Figure 5 As shown, serum GnRH, serum LH, and serum and testicular TE levels were significantly increased in LOH mice treated with mixed fermented milk. Figure 5Given that GnRH and LH are upstream regulators of testicular TE synthesis, and that they maintain physiological testosterone levels through the pulsatile secretion pattern of the HPG axis, it can be concluded that the increase in testicular TE levels induced by mixed fermented milk is achieved through regulation of the HPG axis. Furthermore, the mixed fermented milk intervention in LOH mice showed an increasing trend in the expression of Hsd17b1, a key gene in the testicular TE synthesis pathway, and a significant increase in the expression of Star, Hsd3b1, and P450scc. Figure 5 The EF results corroborate the fact that testicular TE levels are increased. In conclusion, mixed fermented milk significantly improves testicular TE synthesis levels in male LOH mice, which is achieved through regulation of the HPG axis.

[0065] 8. The effect of mixed fermented milk on male sperm quality

[0066] After the experiment, mice in each group were anesthetized and euthanized using isoflurane. The right testis of each mouse was removed, and the sac was removed. Two portions (50 mg each) were taken and placed into two test tubes containing Medium 199 (Thermofisher, serial number: 11150059), and incubated at 37°C for 2 hours. Subsequently, the sperm were stained with DIFF and subjected to morphological analysis. Then, computer-aided sperm analysis (CASAA) was used to detect routine semen parameters and motility parameters.

[0067] The results are as follows Figure 6 As shown, morphological analysis revealed that the number of abnormal sperm in LOH mice treated with mixed fermented milk was significantly lower than that in model LOH mice. Figure 6 AB). Preliminary CASA analysis showed that mixed fermented milk intervention could significantly improve multiple characteristics and motility parameters of sperm in LOH mice, including 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, and average whiplash frequency of sperm. Figure 6 In conclusion, mixed fermented milk significantly improved sperm quality in male LOH mice.

[0068] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0069] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0070] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0071] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. A compound microbial agent comprising Lactobacillus rhamnosus and Streptococcus thermophilus, wherein the Lactobacillus rhamnosus has the preservation number CGMCC No. 13310 and the Streptococcus thermophilus has the preservation number CGMCC No. 17323.

2. The compound microbial agent as described in claim 1, characterized in that, The effective viable count ratio of *Lactobacillus rhamnosus* to *Streptococcus thermophilus* is (80-95):(0.5-1), preferably, the effective viable count ratio of *Lactobacillus rhamnosus* to *Streptococcus thermophilus* is 92.5:0.

8.

3. A fermented milk, prepared by fermentation using the compound microbial agent described in claim 1.

4. The fermented milk as described in claim 2, characterized in that, The fermented milk also includes an arginine solution. Preferably, the concentration of arginine in the fermented milk is 0.1% to 0.5% g / mL, and more preferably, the concentration of arginine in the fermented milk is 0.25% g / mL.

5. The fermented milk as described in claim 3, characterized in that, The arginine solution is an aqueous solution of arginine.

6. The use of the compound microbial agent as described in claim 1 or 2, or the fermented milk as described in any one of claims 3 to 5, in the preparation of products for treating delayed-onset hypogonadism.

7. The application as described in claim 6, characterized in that, The product enhances male sexual function; And / or, the product improves male fertility; And / or, the product improves male reproductive capacity, preferably, the product increases the rate of conception in female individuals and enhances the reproductive potential of female individuals; And / or, 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. And / or, the product improves the oxidative stress activation of male testes; preferably, the product increases the activity of testicular superoxide dismutase or catalase, reduces testicular malondialdehyde levels, and increases the transcriptional level of antioxidant genes Prx3, Prx4, Gstm5, or Gpx4 in the testes.

8. The application as described in claim 6, 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 enhances the expression of genes Star, Hsd3b1, Hsd17b1, and P450scc in the testicular testosterone synthesis pathway.

9. The application as described in claim 6, 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 6, characterized in that, The products include any one or more of the following: food, medicine, or health products.

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