Fermented product of Bifidobacterium longum subsp. infantis, preparation method thereof, and application thereof in increasing testosterone level and fertilization rate, improving chronic kidney disease, and resisting fatigue

Abstract

The present invention belongs to the field of probiotic fermentation technology, and specifically relates to a fermented product of Bifidobacterium longum subsp. infantis, and a preparation method thereof, and applications thereof in improving testosterone levels and fertilization rates, improving chronic kidney disease, and combating fatigue. The Bifidobacterium longum subsp. infantis NKU FB3-14 composition of the present invention and a probiotic fermented product prepared using the Bifidobacterium longum subsp. infantis NKU FB3-14 composition as a raw material have bioactive substances that are different from the raw materials, thereby achieving the effects of improving testosterone levels and fertilization rates, improving chronic kidney disease, and combating fatigue.

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotic fermentation, and particularly relates to a fermentation product of Bifidobacterium longum subspecies infantis, a preparation method thereof, and applications in increasing testosterone levels and fertilization rates, improving chronic kidney disease, and resisting fatigue. Background Art

[0002] With the development of modern society, people's work and life paces are gradually accelerating, resulting in increased stress and sub-healthy diets, and as a result, more and more people are suffering from fatigue. Fatigue is a complex physiological phenomenon caused by the interaction of multiple factors and multiple systems. It refers to the maladaptation caused by "the body's inability to maintain a specific level of function or the overall inability to maintain a predetermined exercise intensity." Energy balance is disrupted during exercise, and the energy supply to the central nervous system is affected, which is a major cause of fatigue in the body. If the body is in a state of fatigue for a long time, it will cause symptoms such as spleen and stomach weakness, metabolic system problems, and even lead to various secondary diseases such as functional disorders, which are harmful to human health.

[0003] While ideal methods for combating fatigue remain elusive, fermented products offer certain advantages. For example, fermented broths of compound Chinese herbal medicines contain a variety of bioactive substances with multiple targets. These compounds can alleviate fatigue by increasing energy, inhibiting central nervous system fatigue, and clearing accumulated metabolites, effectively regulating physiological functions. Due to their diverse anti-fatigue mechanisms, minimal side effects, and absence of harmful stimulants, fermented foods containing compound Chinese herbal medicines have become a hot topic in anti-fatigue research in recent years.

[0004] Bifidobacterium longum subsp. infantis NKU FB3-14 was isolated from infant fecal samples. Studies have shown that it exhibits promising anti-inflammatory and antioxidant effects, regulates intestinal flora, alleviates constipation, and improves liver metabolism. In recent years, the use of probiotic fermentation broths in the development of sports health foods and / or supplements has gained increasing attention. However, few studies have reported on the effects of using Bifidobacterium longum subsp. infantis in nutritional fermentation broths containing compound Chinese herbal medicines to increase testosterone levels and fertilization rates, improve chronic kidney disease, and combat fatigue. Summary of the Invention

[0005] The present invention aims to provide a fermentation product of Bifidobacterium longum subsp. infantis, a preparation method thereof, and an application thereof in improving testosterone levels and fertilization rates, improving chronic kidney disease, and resisting fatigue. The fermentation product of Bifidobacterium longum subsp. infantis NKU FB3-14 has the effects of improving testosterone levels and fertilization rates, improving chronic kidney disease, and resisting fatigue.

[0006] The present invention provides a Bifidobacterium longum subsp. infantis ( Bifidobacterium longum subsp. infantis ) NKU FB3-14 composition, comprising raw materials and Bifidobacterium longum infantis subsp. NKU FB3-14; the raw materials comprise, by weight, 25-35 parts of Cistanche deserticola, 10-20 parts of ginseng, 5-15 parts of raspberry, 5-10 parts of cinnamon bark, 5-15 parts of Poria cocos, 5-10 parts of wolfberry, 10-15 parts of Chinese yam, 5-10 parts of Polygonatum sibiricum, 5-10 parts of Eucommia ulmoides male flowers, and 10-20 parts of oyster; the concentration of Bifidobacterium longum infantis subsp. NKU FB3-14 in the Bifidobacterium longum infantis subsp. NKU FB3-14 composition is 1×10 7 ~1×10 9 CFU / mL; the preservation number of the Bifidobacterium longum subspecies infantis NKU FB3-14 is CGMCC No.25762, and the preservation date is September 21, 2022.

[0007] Preferably, the raw materials include: 30 parts of Cistanche deserticola, 15 parts of ginseng, 10 parts of raspberry, 10 parts of cinnamon, 10 parts of Poria cocos, 10 parts of wolfberry, 10 parts of yam, 10 parts of polygonatum, 10 parts of eucommia ulmoides flowers and 10 parts of oyster.

[0008] The present invention also provides a fermentation product of Bifidobacterium longum subsp. infantis NKU FB3-14, which is prepared by fermenting the Bifidobacterium longum subsp. infantis NKU FB3-14 composition described in the above scheme.

[0009] The present invention also provides a preparation method of the fermentation product of Bifidobacterium longum subspecies infantis NKU FB3-14 described in the above scheme, comprising the following steps: mixing raw materials and water, and then soaking and decocting in sequence to obtain a water extract; concentrating the water extract to a crude drug amount of 1 g / mL to obtain an extract; or, crushing the raw materials and mixing them with water to a crude drug amount of 1 g / mL to obtain a mixture; inoculating Bifidobacterium longum subspecies infantis NKU FB3-14 into the extract or the mixture for fermentation to obtain the fermentation product of Bifidobacterium longum subspecies infantis NKU FB3-14.

[0010] Preferably, the OD of the Bifidobacterium longum subspecies infantis NKU FB3-14 is 600 The value is 2; the inoculation amount of the Bifidobacterium longum subspecies infantis NKU FB3-14 is 9% to 11% of the volume of the extract.

[0011] Preferably, the fermentation time is 10-24 hours and the temperature is 36-38°C.

[0012] The present invention also provides the use of the Bifidobacterium longum subsp. infantis NKU FB3-14 composition described in the above scheme or the fermentation product of Bifidobacterium longum subsp. infantis NKU FB3-14 or the fermentation product of Bifidobacterium longum subsp. infantis NKU FB3-14 prepared by the preparation method in food.

[0013] The present invention also provides the use of the Bifidobacterium longum subsp. infantis NKU FB3-14 composition described in the above scheme, or the fermentation product of Bifidobacterium longum subsp. infantis NKU FB3-14, or the fermentation product of Bifidobacterium longum subsp. infantis NKU FB3-14 prepared by the preparation method in the preparation of a product having one or more of the following functions, wherein the product is a non-food product:

[0014] 1) Improve body muscle tissue damage; 2) Improve exercise capacity; 3) Improve the body's energy metabolism level; 4) Improve the body's endurance; 5) Improve chronic kidney disease; 6) Improve fatigue caused by reproductive factors.

[0015] Preferably, the improvement of chronic kidney disease includes improving kidney tissue damage and / or reducing the level of kidney tissue damage markers; the kidney tissue damage markers include blood creatinine; and the improvement of fatigue caused by reproductive factors includes one or more of increasing testosterone levels in testicular cells, reducing oxidative stress levels in testicular cells, improving testicular tissue damage, improving androgen levels, and increasing fertilization rate.

[0016] Beneficial effects:

[0017] The present invention provides a Bifidobacterium longum infantis subspecies NKU FB3-14 composition, comprising raw materials and Bifidobacterium longum infantis subspecies NKU FB3-14; the raw materials comprise, by weight, 25-35 parts of Cistanche deserticola, 10-20 parts of ginseng, 5-15 parts of raspberry, 5-10 parts of cinnamon bark, 5-15 parts of Poria cocos, 5-10 parts of wolfberry, 10-15 parts of Chinese yam, 5-10 parts of Polygonatum sibiricum, 5-10 parts of Eucommia ulmoides male flower, and 10-20 parts of oyster; the concentration of Bifidobacterium longum infantis subspecies NKU FB3-14 in the Bifidobacterium longum infantis subspecies NKU FB3-14 composition is 1×10 7 ~1×10 9 CFU / mL; the deposit number of the Bifidobacterium longum subsp. infantis NKU FB3-14 is CGMCC No. 25762. The Bifidobacterium longum subsp. infantis NKU FB3-14 composition of the present invention and the fermentation product prepared using the Bifidobacterium longum subsp. infantis NKU FB3-14 composition as a raw material have different bioactive substances from the raw materials, thereby achieving the effects of increasing testosterone levels and fertilization rates, improving chronic kidney disease, and resisting fatigue. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0019] Figure 1 This is a growth curve of Bifidobacterium longum subsp. infantis NKU FB3-14 in Example 1 in the extract and BS liquid culture medium;

[0020] Figure 2 This is a technical roadmap for metabolomics analysis of the fermentation broth of Bifidobacterium longum subsp. infantis NKU FB3-14 in Example 1;

[0021] Figure 3 This is a circular diagram of the metabolic class composition of the fermentation broth of Bifidobacterium longum subsp. infantis NKU FB3-14 in Example 1;

[0022] Figure 4 This is a PCA analysis result diagram of the fermentation broth of Bifidobacterium longum subsp. infantis NKU FB3-14 in Example 1;

[0023] Figure 5 This is a volcano plot of the differential metabolite analysis of Bifidobacterium longum subsp. infantis NKU FB3-14 before and after fermentation in the raw material in Example 1;

[0024] Figure 6 This is a volcano plot of differential metabolite analysis of Bifidobacterium longum subsp. infantis NKU FB3-14 in Example 1 after fermentation in the raw material for 18 h and 24 h;

[0025] Figure 7 This is a graph showing the difference in amino acid content of Bifidobacterium longum subsp. infantis NKU FB3-14 in Example 1 after fermentation in the raw material for 18 hours and 24 hours;

[0026] Figure 8 This is a graph showing the difference in bile acid content of Bifidobacterium longum subsp. infantis NKU FB3-14 in Example 1 after fermentation in the raw material for 18 hours and 24 hours;

[0027] Figure 9 This is a graph showing the difference in flavonoid content of Bifidobacterium longum subsp. infantis NKU FB3-14 in Example 1 after fermentation in the raw material for 18 hours and 24 hours;

[0028] Figure 10 This is a graph showing the difference in coenzyme and vitamin content after fermentation of Bifidobacterium longum subsp. infantis NKU FB3-14 in the raw material for 18 hours and 24 hours in Example 1;

[0029] Figure 11This is a graph showing the difference in terpenoid content after fermentation of Bifidobacterium longum subsp. infantis NKU FB3-14 in the raw material for 18 hours and 24 hours in Example 1;

[0030] Figure 12 This is a graph showing the difference in steroid compound content in Bifidobacterium longum subsp. infantis NKU FB3-14 fermented in the raw material for 18 hours and 24 hours in Example 1;

[0031] Figure 13 This is a graph showing the effects of seven groups of fermentation broths on the proliferation rate of MLTC-1 cells in Example 2;

[0032] Figure 14 This is a graph showing the effects of the seven fermentation broths on the testosterone concentration in the supernatant of MLTC-1 cells in Example 2;

[0033] Figure 15 Figure 3 is the RNA expression graph of oxidative stress level in testicular cells of each group;

[0034] Figure 16 Graph showing the testosterone content in testicular cells of each group in Example 3;

[0035] Figure 17 The behavioral trajectory path map and behavioral trajectory heat map of each group of zebrafish in Example 4;

[0036] Figure 18 This is a statistical result diagram of the total movement distance of each group of zebrafish in Example 4;

[0037] Figure 19 HE staining of muscle tissue pathological sections of zebrafish in each group in Example 4;

[0038] Figure 20 This is a statistical result diagram of ATP content, creatine kinase content and lactic acid content in the muscle of each group of zebrafish in Example 4;

[0039] Figure 21 HE staining of testicular tissue pathological sections of each group of zebrafish in Example 4;

[0040] Figure 22 This is a statistical result diagram of the male hormone levels of each group of zebrafish in Example 4;

[0041] Figure 23 HE staining of kidney tissue pathological sections of zebrafish in each group in Example 4;

[0042] Figure 24 This is a statistical graph of the blood creatinine levels of each group of zebrafish in Example 4;

[0043] Figure 25Graph showing the statistical results of the total number of eggs, number of fertilized eggs, and fertilization rate after mating of each group of zebrafish in Example 4. DETAILED DESCRIPTION

[0044] The present invention provides a Bifidobacterium longum infantis subspecies NKU FB3-14 composition, comprising raw materials and Bifidobacterium longum infantis subspecies NKU FB3-14; the raw materials comprise, by weight, 25-35 parts of Cistanche deserticola, 10-20 parts of ginseng, 5-15 parts of raspberry, 5-10 parts of cinnamon bark, 5-15 parts of Poria cocos, 5-10 parts of wolfberry, 10-15 parts of Chinese yam, 5-10 parts of Polygonatum sibiricum, 5-10 parts of Eucommia ulmoides male flower, and 10-20 parts of oyster; the concentration of Bifidobacterium longum infantis subspecies NKU FB3-14 in the Bifidobacterium longum infantis subspecies NKU FB3-14 composition is 1×10 7 ~1×10 9 CFU / mL; the deposit number of the Bifidobacterium longum subspecies infantis NKU FB3-14 is CGMCC No.25762.

[0045] In the present invention, the Bifidobacterium longum subsp. infantis NKU FB3-14 has been disclosed in Chinese patent CN116286551A, and experiments have shown that the Bifidobacterium longum subsp. infantis NKU FB3-14 is a safe strain that can be used in humans and animals.

[0046] In one embodiment, the mass fraction of Cistanche deserticola in the Bifidobacterium longum subsp. infantis NKU FB3-14 composition of the present invention is 28-32 parts; in another embodiment, the mass fraction of Cistanche deserticola in the Bifidobacterium longum subsp. infantis NKU FB3-14 composition of the present invention is 30 parts; in another embodiment, the mass fraction of ginseng in the Bifidobacterium longum subsp. infantis NKU FB3-14 composition of the present invention is 12-18 parts; in another embodiment, the mass fraction of ginseng is 15 parts. In another embodiment, the mass fraction of raspberry in the Bifidobacterium longum subsp. infantis NKU FB3-14 composition of the present invention is 10-14 parts. In another embodiment, the mass fraction of cinnamon in the Bifidobacterium longum subsp. infantis NKU FB3-14 composition of the present invention is 8-10 parts. In another embodiment, the mass fraction of Poria cocos in the Bifidobacterium longum subsp. infantis NKU FB3-14 composition of the present invention is 8-10 parts. As an embodiment, the mass fraction of wolfberry in the Bifidobacterium longum subsp. infantis NKU FB3-14 composition of the present invention is 8 to 10 parts. As an embodiment, the mass fraction of Chinese yam in the Bifidobacterium longum subsp. infantis NKU FB3-14 composition of the present invention is 10 to 12 parts. As an embodiment, the mass fraction of polygonatum in the Bifidobacterium longum subsp. infantis NKU FB3-14 composition of the present invention is 8 to 10 parts. As an embodiment, the mass fraction of eucommia male flower in the Bifidobacterium longum subsp. infantis NKU FB3-14 composition of the present invention is 8 to 10 parts. As an embodiment, the mass fraction of oyster in the Bifidobacterium longum subsp. infantis NKU FB3-14 composition of the present invention is 10 to 15 parts.

[0047] The present invention also provides a fermented product of Bifidobacterium longum subsp. infantis NKU FB3-14, which is prepared by fermenting the Bifidobacterium longum subsp. infantis NKU FB3-14 composition described in the above technical solution. As an embodiment, the dosage form of the fermented product of Bifidobacterium longum subsp. infantis NKU FB3-14 of the present invention can be, but is not limited to, a liquid and / or a powder.

[0048] Specifically, the present invention also provides a preparation method for the fermentation product of Bifidobacterium longum subspecies infantis NKU FB3-14 described in the above technical solution, comprising the following steps: mixing the raw materials and water, soaking and decocting in sequence to obtain a water extract; concentrating the water extract to a crude drug amount of 1 g / mL to obtain an extract; or, crushing the raw materials and mixing them with water to a crude drug amount of 1 g / mL to obtain a mixture; inoculating Bifidobacterium longum subspecies infantis NKU FB3-14 into the extract or the mixture for fermentation to obtain the Bifidobacterium longum subspecies infantis NKU FB3-14 fermentation product.

[0049] The present invention mixes raw materials and water and then soaks and decocts them in sequence to obtain a water extract. The present invention has no special requirements for the soaking and decoction methods, and methods well known to those skilled in the art can be used.

[0050] After obtaining the water extract, the present invention concentrates the water extract to a crude drug amount of 1 g / mL to obtain an extract. As an embodiment, the concentration method can be reduced pressure concentration.

[0051] Alternatively, the present invention comprises pulverizing the raw material and mixing it with water until the crude drug amount is 1 g / mL to obtain a mixture. In one embodiment, the pulverization method may be grinding the raw material into a powder. In another embodiment, the pulverization method may be freeze-drying the raw material and then grinding it into a powder.

[0052] After obtaining the extract or mixture, the present invention inoculates Bifidobacterium longum subspecies infantis NKU FB3-14 into the extract or mixture for fermentation to obtain the fermentation product of Bifidobacterium longum subspecies infantis NKU FB3-14. As an embodiment, during the inoculation, the OD value of Bifidobacterium longum subspecies infantis NKU FB3-14 is 600The value is 2. In one embodiment, the inoculum size of Bifidobacterium longum subsp. infantis NKU FB3-14 is 9% to 11% of the volume of the extract; in another embodiment, the inoculum size of Bifidobacterium longum subsp. infantis NKU FB3-14 is 10% of the volume of the extract. In one embodiment, during the inoculation, the Bifidobacterium longum subsp. infantis NKU FB3-14 is inoculated in the form of a bacterial liquid of Bifidobacterium longum subsp. infantis NKU FB3-14. In one embodiment, the preparation method of the bacterial liquid of Bifidobacterium longum subsp. infantis NKU FB3-14 comprises: inoculating Bifidobacterium longum subsp. infantis NKU FB3-14 into BS liquid culture medium for fermentation to obtain the bacterial liquid of Bifidobacterium longum subsp. infantis NKU FB3-14. In one embodiment, the fermentation temperature is 36-38°C; in another embodiment, the fermentation temperature is 37°C. As one embodiment, the fermentation culture time is 36 to 48 hours; as another embodiment, the fermentation time is 40 to 44 hours, that is, the Bifidobacterium longum subsp. infantis NKU FB3-14 is fermented to the logarithmic phase under the above-mentioned fermentation culture conditions, and then the obtained Bifidobacterium longum subsp. infantis NKU FB3-14 bacterial liquid is inoculated into the extract.

[0053] In one embodiment, during the fermentation process of inoculating Bifidobacterium longum subsp. infantis NKU FB3-14 into the extract, the fermentation time is 10 to 24 hours; in another embodiment, the fermentation time is 18 to 24 hours. In one embodiment, the fermentation temperature is 36 to 38°C; in another embodiment, the fermentation culture temperature is 37°C.

[0054] The present invention also provides the use of the Bifidobacterium longum subsp. infantis NKU FB3-14 composition described in the above technical solution, the Bifidobacterium longum subsp. infantis NKU FB3-14 fermentation product described in the above technical solution, or the Bifidobacterium longum subsp. infantis NKU FB3-14 fermentation product prepared by the preparation method described in the above technical solution in food.

[0055] As an embodiment, the food may be an anti-fatigue food.

[0056] The present invention also provides the use of the Bifidobacterium longum subsp. infantis NKU FB3-14 composition described in the above scheme, or the fermentation product of Bifidobacterium longum subsp. infantis NKU FB3-14, or the fermentation product of Bifidobacterium longum subsp. infantis NKU FB3-14 prepared by the preparation method in the preparation of a product having one or more of the following functions, wherein the product is a non-food product:

[0057] 1) Improve body muscle tissue damage; 2) Improve exercise capacity; 3) Improve the body's energy metabolism level; 4) Improve the body's endurance; 5) Improve chronic kidney disease; 6) Improve fatigue caused by reproductive factors.

[0058] As an embodiment, the product may be a medicine.

[0059] In one embodiment, the improvement of body endurance may be achieved by increasing the level of muscle capacity markers; in another embodiment, the improvement of muscle capacity marker levels may be achieved by increasing one or more of ATP content, creatine kinase content, and lactic acid content.

[0060] As an embodiment, the improvement of fatigue caused by chronic kidney disease factors includes improving kidney tissue damage and / or reducing the level of kidney tissue damage markers; the kidney tissue damage markers include blood creatinine.

[0061] As an embodiment, the improvement of fatigue caused by reproductive factors includes one or more of increasing the testosterone level in testicular cells, reducing the oxidative stress level in testicular cells, improving testicular tissue damage, improving androgen levels, and increasing fertilization rate.

[0062] In one embodiment, the improvement of testicular tissue damage can be improving testicular tissue pathological damage and / or increasing androgen levels. In another embodiment, the improvement of androgen levels can be increasing luteinizing hormone content and / or increasing testosterone content.

[0063] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0064] The components of the culture medium used in the following examples are as follows:

[0065] BS liquid medium: peptone 10.0 g / L, liver extract powder 5.0 g / L, beef extract powder 3.0 g / L, yeast extract powder 5.0 g / L, trypticase peptone 8.0 g / L, soluble starch 0.5 g / L, sodium chloride 1.0 g / L, dipotassium hydrogen phosphate 1.0 g / L, potassium dihydrogen phosphate 1.0 g / L, FeSO4·7H2O 0.3 g / L, L-cysteine ​​0.5 g / L, MnSO4 0.005 g / L, and glucose 10.0 g / L.

[0066] In the following examples, statistical significance was determined by Student's t The significance level is set to * p (or* p ) < 0.05, ** p (or** p) < 0.01, *** p (or*** p ) < 0.001, **** p (or**** p )<0.0001.

[0067] Example 1

[0068] 1. Raw material ratio and preparation method of Bifidobacterium longum subsp. infantis NKU FB3-14 fermentation product:

[0069] Ingredients: Cistanche deserticola 30g, ginseng 15g, raspberry 10g, cinnamon 10g, Poria 10g, wolfberry 10g, Chinese yam 10g, polygonatum 10g, eucommia male flower 10g and oyster 10g;

[0070] The raw materials and water are mixed and then soaked and decocted in sequence to obtain a water extract; the water extract is concentrated under reduced pressure to a crude drug amount of 1 g / mL to obtain an extract; the raw materials are freeze-dried and then ground into powder to obtain freeze-dried powder for later use.

[0071] The bacterial liquid of Bifidobacterium longum subsp. infantis NKU FB3-14 (OD 600 The extract (or the extract obtained by dissolving the lyophilized powder in water to 1 g / mL) and BS liquid culture medium were inoculated at 10% of the volume, and fermented at 37°C. The growth curves of the two fermentation groups were observed. The results are shown in the figure. Figure 1 As shown, in Figure 1 The fermentation broth group and BS medium group represent the growth curves of Bifidobacterium longum subsp. infantis NKU FB3-14 fermented in the extract and BS liquid medium, respectively.

[0072] Depend on Figure 1 It can be concluded that compared with BS liquid culture medium, the growth of Bifidobacterium longum subsp. infantis NKU FB3-14 in the extract environment is not as good as that of Bifidobacterium longum subsp. infantis NKU FB3-14 in BS liquid culture medium, but the growth trend is consistent with the growth trend of bacteria, which shows that the live bacteria can survive in the extract for 24 hours.

[0073] 2. Ingredient Identification: Changes in Formula Composition After Fermentation with Bifidobacterium longum subsp. infantis NKU FB3-14

[0074] In order to study the dynamic changes of the chemical composition of the extract, this example uses liquid chromatography-mass spectrometry (LC-MS) to perform non-targeted metabolome detection and analysis. Specifically, the fermentation broth of Bifidobacterium longum subspecies infantis NKU FB3-14 in step 1 before fermentation of the extract (i.e., fermentation 0h), 10h after fermentation, 18h after fermentation, and 24h after fermentation is subjected to solid-liquid separation, the bacterial suspension is centrifuged and passed through a membrane, and the obtained supernatant is processed for non-targeted metabolome analysis. Each group has three biological replicates, and the four groups are respectively recorded as FTCM0, FTCM10, FTCM18, and FTCM24. The samples of the four groups are qualitatively analyzed by LC-MS to qualitatively measure the types of substances, and the analysis data are sorted and analyzed for principal component analysis (PCA), differential metabolites, and volcano plots. The detection technology roadmap is shown in FIG. Figure 2 shown.

[0075] 2.1 Metabolite category composition ring diagram Figure 3 As shown in the table, the overall expression of metabolites in each sample is aggregated, and the composition of metabolites has sample diversity. After fermentation, there will be different categories of substances, among which alcohol and amine substances account for about 3.76%, aldehydes, ketones and esters account for 6.8%, alkaloids account for 1.33%, amino acids and their metabolites account for 15.3%, benzene and its derivatives account for 17.93%, bile acid account for 0.38%, carbohydrates and their metabolites account for 2.53%, coenzymes and vitamins account for 0.57%, and fatty acids account for 4.7%. 4%, flavonoids accounted for 2.75%, glucose accounted for 0.19%, glycoproteins accounted for 1.49%, heterocyclic compounds accounted for 12.99%, hormones and hormone-related compounds accounted for 2.69%, lignin and coumarin substances accounted for 0.41%, nucleotides and metabolites accounted for 3.22%, organic compound machine derivatives accounted for 13.53%, phenolic acid accounted for 0.41%, sphingomyelin accounted for 0.09%, steroids accounted for 0.66%, terpenes accounted for 0.16%, and choline substances accounted for 2.21%.

[0076] 2.2 PCA analysis results are as follows Figure 4 As shown in the figure, there are obvious differences between samples at different fermentation stages. 71% of the features are shared between samples. Each group has a strong clustering tendency and is clearly separated from each other. There is a clear classification at 0h of fermentation and at other times, and the similarity within the same group is high.

[0077] 2.3 Analysis of the differential metabolites in the FTCM18 and FTCM0 samples. The results are as follows Figure 5 As shown, 1064 differential metabolites were significantly upregulated and 112 differential metabolites were downregulated compared with 0 h after fermentation 18 h. The metabolites produced 18 h after fermentation and 0 h after fermentation were quite different.

[0078] 2.4 The metabolites in the fermentation broth were compared using FTCM0 as the control group and FTCM18 as the fermentation focus. The results are shown in Tables 1 and Figure 5 As shown in Table 1 and Figure 5 It was concluded that after 18 hours of fermentation, the content of several small peptides, polypeptides, and proteins increased: 1) It significantly increased the content of prostaglandin A1, a dehydrated product of PGE1 originally isolated from human semen. It is an anti-inflammatory cyclopentenone prostaglandin that can cause renal vasodilation, increase urinary sodium excretion, and lower arterial pressure in hypertensive patients. 2) It also increased the content of several peptides and proteins, and elevated the levels of natural hormones, which play important regulatory roles in metabolism, growth, development, reproduction, sex, libido, and sexual activity. 3) It also increased the amount of pyridoxal, which can be widely used in digestive tract diseases, peripheral neuritis caused by food-drug interactions, insomnia, leukopenia, and the treatment of infantile seizures or for pregnant women to prevent them. 4) It also increased the level of serotonin, which is responsible for transmitting signals from one part of the brain to another—playing a significant role in consciousness, attention, cognition, and emotion, and also regulates other systems in the body. Serotonin is often thought of as a neurotransmitter, responsible for transmitting information between neurons. However, serotonin also has a secondary role as a chemical in the body: a hormone released into the bloodstream. It can send messages to various parts of the body, including the intestines and platelets. Microbes can be used to enhance the metabolism of functional compounds.

[0079] Table 1 Changes in main substances

[0080]

[0081] 2.5 Analysis of the differential metabolites in the FTCM18 and FTCM24 groups of samples. The results are as follows Figure 6 As shown, there is no significant difference in the metabolites 18h after fermentation and 24h after fermentation. There are 6460 metabolites with no significant difference, indicating that there is no significant change in the metabolites 18h after fermentation and 24h after fermentation, proving that the substances produced by fermentation 18h and 24h are similar, so 18h is determined as the fermentation time.

[0082] The present invention ferments the fermented food strain Bifidobacterium longum infantis subspecies NKU FB3-14 in the raw material, and comprehensively analyzes the changes of metabolites before and after fermentation. Figure 5 Results from the study found that the fermented broth significantly increased the amount of hormones or hormone-like products, potentially contributing to antioxidant activity and fatigue relief. These changes suggest that fermentation with Bifidobacterium longum subsp. infantis NKU FB3-14 alters the active ingredients in the raw material.

[0083] 2.6 The differences in amino acids, bile acids, flavonoids, coenzymes and vitamins, terpenoids and steroids before fermentation (FTCM0) and after fermentation (FTCM18) were analyzed. Figures 7-12 It can be seen that compared with those before fermentation, the amounts of amino acids, bile acids, flavonoids, coenzymes and vitamins, terpenoids and steroid compounds in the fermentation broth after fermentation increased.

[0084] Therefore, fermentation of Bifidobacterium longum subsp. infantis NKU FB3-14 can be used as an effective processing method to improve the biological functions of raw materials.

[0085] Example 2

[0086] The preparation method in step 1 of Example 1 was used; the raw material formulas were as follows:

[0087] 0% Cistanche deserticola: Compared with the formula in Example 1, the amount of Cistanche deserticola added is 0g, and the amounts of other components remain unchanged;

[0088] 30% Cistanche deserticola: Compared with the formula in Example 1, the amount of Cistanche deserticola added is 9 g, and the amounts of other components remain unchanged;

[0089] 50% Cistanche deserticola: Compared with the formula in Example 1, the amount of Cistanche deserticola added is 15g, and the amounts of other components remain unchanged;

[0090] 0% ginseng: compared with the formula in Example 1, the amount of ginseng added is 0g, and the amounts of other components remain unchanged;

[0091] 30% ginseng: compared with the formula in Example 1, the amount of ginseng added is 4.5 g, and the amounts of other components remain unchanged;

[0092] 50% ginseng: compared with the formula in Example 1, the added amount of ginseng is 7.5 g, and the amounts of other components remain unchanged.

[0093] The samples were prepared using the above six groups and the fermentation product of Bifidobacterium longum subsp. infantis NKU FB3-14 prepared in step 1 of Example 1 (referred to as the whole formula). After filtering through a 0.22 μm filter, the samples were added to a six-well plate at a ratio of 20% of the total volume of the cell culture medium. The cells were then treated with mouse Leydig cell tumor MLTC-1. The effects of the seven groups of fermentation broth on the proliferation rate of MLTC-1 cells and the testosterone concentration in the cell supernatant were evaluated. The results are shown in FIG. Figures 13 and 14 shown.

[0094] Depend on Figures 13 and 14 It can be concluded that compared with each reduced-dose group, the full-prescription group has the most significant promoting effect on the proliferation rate of MLTC-1 cells and the testosterone concentration in the cell supernatant.

[0095] Example 3

[0096] In vivo experiments using mouse testicular Leydig cell tumor cells MLTC-1 were used to verify the efficacy of the fermentation product of Bifidobacterium longum subsp. infantis NKUFB3-14 in alleviating mouse testicular Leydig cell tumor cell damage and oxidative stress

[0097] 1. Experimental Design

[0098] After freezing, MLTC-1 cells were revived and cultured until the cell density increased to more than 80%. MLTC-1 cells were placed in a 37°C, 5% CO2 cell culture incubator containing RPMI-1640 + 10% FBS + 1% P / S basal culture medium for subculture, and cells in the logarithmic growth phase were selected for experiments.

[0099] Based on preliminary experimental results, MLTC-1 cells were divided into four groups: a blank control group (complete medium only); a model control group (denoted as H2O2) (100μM H2O2); a water extract group (denoted as TCM) (100μM H2O2 + 20% TCM); and a fermentation broth group (denoted as FTCM) (100μM H2O2 + 20% FTCM). Subsequent cell experiments were performed after 24 hours of culture. TCM represents the extract prepared in step 1 of Example 1, and FTCM refers to the fermentation product of Bifidobacterium longum subsp. infantis NKU FB3-14, fermented for 18 hours in Example 1.

[0100] 2. Determination of Oxidative Stress Levels

[0101] After the cells were grouped and treated, the culture medium was removed and the cells were washed with PBS. The total RNA was extracted using TRIzol reagent and its concentration was determined. The RNA was reverse transcribed into cDNA for subsequent amplification. PCR amplification conditions: stage 1, pre-denaturation at 90°C for 30 seconds; stage 2 (40 cycles), denaturation at 95°C for 15 seconds, annealing and extension at 60°C for 30 seconds; stage 3 (melting curve), 65°C → 95°C, with collection every 0.5°C increase in temperature. 2 －ΔΔCt Legal analysis, GAPDH The relative expression levels of oxidative stress-related gene mRNA in each group were determined using the internal reference gene. The primer sequences are shown in Table 2. Figure 15 As shown. (# is compared with TCM p <0.05, ## is compared with TCM p <0.01, **compared with H2O2 p <0.01).

[0102] Table 2 Primer sequences

[0103]

[0104] Depend on Figure 15 It can be concluded that compared with the model control group, the water extract group and the fermentation broth group both upregulated the levels of steroidogenic acute regulatory protein (STAR) and luteinizing hormone receptor (LHR); upregulated the CYP450 enzymes that catalyze many reactions related to the metabolism and synthesis of cholesterol, steroids and other lipids ( CYP11A1 and CYP17A1 The levels of 17β-HSDs (17β-HSDs) were significantly increased in both the water extract and fermentation broth groups, and the intervention effect in the fermented broth group was superior to that in the unfermented water extract group.

[0105] 3. Determination of testosterone levels

[0106] After the cells were grouped and treated, the culture medium was removed, the cells were digested with trypsin, and the cells were collected by centrifugation; the collected cells were washed three times with cold PBS; an appropriate amount of PBS was taken to resuspend the cells, and the cell suspension was treated with ultrasound in an ice bath at a power of 60 Hz for 60 s to cause the cells to be rapidly shaken and broken; the mixture after shaking and breaking was centrifuged at 2-8°C and 1500×g for 10 min, and the supernatant was collected for later use.

[0107] The results are as follows Figure 16 As shown (*compared with H2O2), it can be seen that both the fermentation liquid group and the water extract group can enhance the effect of H2O2-induced decrease in testosterone content in testicular cells.

[0108] Example 4

[0109] In vivo zebrafish experiments verify the application of Bifidobacterium longum subsp. infantis NKU FB3-14 fermentation preparation in anti-fatigue, chronic kidney disease intervention and kidney nourishment

[0110] 1. Experimental Design and Sample Collection

[0111] AB wild zebrafish were raised in the zebrafish culture system of Nankai University School of Medicine at a constant ambient temperature (28 ± 0.5°C) with a 14 h / 10 h day / night cycle, and embryonic reproduction was carried out by natural pair mating.

[0112] Using a laboratory-verified zebrafish injury model - zebrafish induced by the food thermal processing hazard heterocyclic amine 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), according to previous laboratory research results (https: / / doi.org / 10.1016 / j.jhazmat.2021.125230): Behavioral and molecular biology have confirmed that IQ exposure causes potential neurodegenerative diseases in zebrafish, characterized by fatigue and tissue damage.

[0113] In this example, four-month-old male zebrafish were randomly divided into four groups. A control group (zebrafish were exposed to circulating water from the aquaculture system); a model group (zebrafish were exposed to 80 ng / mL IQ solution); a low-concentration fermentation broth group (or fermentation broth-low concentration group): the fermentation product of Bifidobacterium longum subsp. infantis NKU FB3-14, fermented for 18 hours as prepared in Example 1, was serially diluted 1000-fold to 1 mg / mL using pure water, filtered, and gradiently filtered, based on a crude drug concentration of 1 g / mL. The zebrafish were then exposed to this solution; and a high-concentration fermentation broth group (2 mg / mL, also referred to as the fermentation broth-low concentration group) was exposed to this solution at a concentration twice that of the low-concentration group. Each group had three replicates, with 15 fish per replicate. The exposure solution was changed daily. After 35 days, the zebrafish were euthanized with 0.016% tricaine and dissected on ice. Muscle, testis, kidney, and blood samples from every five fish were combined into a single replicate.

[0114] 2. Zebrafish Behavioral Analysis

[0115] Before the zebrafish were killed, the zebrafish in the control group, model group, low-concentration fermentation liquid group, and high-concentration fermentation liquid group were placed in a Zebra Tower (ViewPoint Life Sciences, Lyon, France) to record their movements. The behavior was evaluated over a 10-minute period using Zebralab ZEB6199 software (ViewPoint Life Sciences). Because fatigue is usually manifested as reduced motor ability and reaction ability, the open field test can be used to intuitively evaluate the motor ability and reaction ability of zebrafish. Next, by tracking the behavioral trajectory of zebrafish and analyzing related parameters, we studied how the fermentation preparation of Bifidobacterium longum subsp. infantis NKU FB3-14 affects the behavior of zebrafish. The results are as follows: Figures 17 and 18 shown.

[0116] Depend on Figure 17It can be seen that the behavioral trajectories and movement heat maps showed that the zebrafish in the control group and the fermentation group showed significantly more movement trajectories and higher activity frequency; on the contrary, the zebrafish in the model group had a fatigue-like reaction, manifested as inactivity, long-term stagnation, fewer movement trajectories and increased stereotyped behavior, indicating that the fermentation preparation of Bifidobacterium longum subspecies infantis NKU FB3-14 can improve motor ability.

[0117] Depend on Figure 18 Analysis of behavioral parameters showed that the total movement distance of zebrafish in the model group was significantly reduced compared to the control group. However, the fermentation solution restored the behavioral changes of zebrafish, once again confirming that the fermentation preparation of Bifidobacterium longum subsp. infantis NKU FB3-14 can increase movement distance, that is, improve the endurance of zebrafish.

[0118] 3. Pathological Analysis of Zebrafish Muscle Tissue HE Staining

[0119] The zebrafish was placed in paraformaldehyde fixative, soaked, and then rinsed overnight. The samples were dehydrated in 70%, 80%, and 90% ethanol solutions in turn for 30 minutes each, then dehydrated twice in 95% ethanol and then twice in 100% ethanol, each for 20 minutes. Placed in a mixture of 1 / 2 volume of pure alcohol and 1 / 2 volume of xylene for 15 minutes, washed with xylene for 5 minutes and then washed with xylene for 15 minutes until transparent. Placed in a mixture of half xylene and half paraffin for 15 minutes, then placed in paraffin I and paraffin II for 55 minutes each, and the transparent agent was removed. The processed samples were embedded, sectioned, spread, baked, HE stained, and sealed. After making muscle tissue pathological sections, the pathological changes in the muscle tissue of zebrafish before and after intervention were observed under an optical microscope to explore the effects of the fermentation preparation of Bifidobacterium longum infantis subspecies NKU FB3-14 on zebrafish muscle tissue damage. The results are as follows: Figure 19 As shown, Figure 19 The scale bar is 25 μm.

[0120] Depend on Figure 19 It can be seen that compared with the control group, the muscle atrophy in the model group was accompanied by inflammatory cell infiltration; while the fermentation group returned to a similar state as the control group, indicating that the fermentation preparation of Bifidobacterium longum subsp. infantis NKU FB3-14 can improve the inflammatory infiltration of muscle tissue.

[0121] At the same time, the ATP content, creatine kinase content, and lactic acid content in zebrafish muscle before and after intervention were measured to verify the effect of Bifidobacterium longum subsp. infantis NKU FB3-14 fermentation preparation on zebrafish muscle tissue damage. ATP content, creatine kinase content, lactic acid content, and total protein concentration were determined using commercial kits (Nanjing Jiancheng Biotechnology Co., Ltd.). The results are as follows: Figure 20 shown.

[0122] Depend on Figure 20 It can be seen that compared with the control group, the ATP content, creatine kinase content and lactic acid content in the muscle of the model group were significantly reduced, indicating that the production of ATP, creatine kinase and lactic acid in the model group was reduced; while the fermentation group increased the content of ATP, creatine kinase and lactic acid to normal levels, indicating that the fermentation preparation of Bifidobacterium longum infantis subsp. NKU FB3-14 can increase the content of ATP, creatine kinase and lactic acid in the muscle, thereby improving physical strength and exerting anti-fatigue function.

[0123] 4. Pathological Analysis of Zebrafish Testis Tissue HE Staining

[0124] After preparing testicular tissue pathological sections, the pathological changes of zebrafish testicular tissue before and after intervention were observed under an optical microscope to explore the effect of Bifidobacterium longum subsp. infantis NKU FB3-14 fermentation preparation on zebrafish testicular tissue damage. The results are as follows Figure 21 As shown, Figure 21 The scale bar is 25 μm.

[0125] Depend on Figure 21 It can be seen that compared with the control group, the area of ​​mature granulosa cells in the testis of the model group was reduced; while the fermentation group returned to a similar state to the control group, indicating that the fermentation preparation of Bifidobacterium longum subsp. infantis NKU FB3-14 can promote the maturation of testicular granulosa cells.

[0126] At the same time, the male hormone levels of zebrafish before and after intervention (luteinizing hormone and testosterone levels in the blood) were tested to verify the effect of the fermentation preparation of Bifidobacterium longum subsp. infantis NKU FB3-14 on zebrafish testicular tissue damage. The luteinizing hormone and testosterone levels were determined using commercial kits (Nanjing Jiancheng Biotechnology Co., Ltd.). The results are as follows: Figure 22 shown.

[0127] Depend on Figure 22 It can be seen that compared with the control group, the luteinizing hormone and testosterone levels in the blood of the model group were significantly reduced; while the fermentation group returned to a similar state to the control group, indicating that the fermentation preparation of Bifidobacterium longum subspecies infantis NKU FB3-14 can increase the level of male hormones.

[0128] 5. Pathological Analysis of Zebrafish Kidney Tissue HE Staining

[0129] After making kidney tissue pathological sections, the pathological changes of zebrafish kidney tissue before and after intervention were observed under an optical microscope to explore the effects of different concentrations of fermentation liquid on zebrafish kidney tissue damage. The results are as follows Figure 23 As shown, Figure 23 The scale bar is 25 μm.

[0130] Depend on Figure 23It can be seen that compared with the control group, the kidneys of the model group were congested and the renal tubules were dilated; while the fermentation group returned to a similar state to the control group, indicating that the fermentation preparation of Bifidobacterium longum subsp. infantis NKU FB3-14 can improve renal tissue congestion and renal tubular dilation.

[0131] At the same time, the blood creatinine levels of zebrafish before and after intervention were detected to verify the effect of Bifidobacterium longum subspecies infantis NKU FB3-14 fermentation preparation on zebrafish kidney tissue damage. Figure 24 As shown. Figure 24 It can be seen that compared with the control group, the creatinine level, a kidney injury marker, in the blood of the model group was significantly increased; while the fermentation group returned to a similar state to the control group, indicating that the fermentation preparation of Bifidobacterium longum subsp. infantis NKU FB3-14 can alleviate kidney tissue damage.

[0132] 6. Analysis of Fertilization Rate after Zebrafish Mating

[0133] Each group of zebrafish were mated with normal female zebrafish at a ratio of 1:1. The number of fertilized eggs and the total number of eggs were calculated to explore the effect of the fermentation preparation of Bifidobacterium longum subsp. infantis NKU FB3-14 on the fertilization rate of zebrafish. The results are as follows Figure 25 shown.

[0134] Depend on Figure 25 It can be seen that compared with the control group, the total number of eggs and the number of fertilized eggs in the model group were significantly reduced, and the fertilization rate was also significantly reduced; while the fermentation group returned to a similar state to the control group, indicating that the fermentation preparation of Bifidobacterium longum infantis subspecies NKU FB3-14 can improve the fertilization rate.

[0135] In summary, fermenting Bifidobacterium longum subsp. infantis NKU FB3-14 in the formula composition provided herein can increase the bioactive substances in the fermented preparation of Bifidobacterium longum subsp. infantis NKU FB3-14, exhibiting anti-fatigue effects. The mechanism of action is related to improving energy metabolism and increasing testosterone levels. The fermented preparation of Bifidobacterium longum subsp. infantis NKU FB3-14 described herein has the effects of increasing testosterone levels and fertilization rates, improving chronic kidney disease, and combating fatigue.

Claims

1. A fermentation product of Bifidobacterium longum subsp. infantis NKU FB3-14, characterized in that The fermentation product of Bifidobacterium longum subspecies infantis NKU FB3-14 is composed of Bifidobacterium longum subspecies infantis ( Bifidobacterium longum subsp. infantis ) NKU FB3-14 composition is prepared by fermentation; the Bifidobacterium longum infantis subspecies NKU FB3-14 composition is composed of raw materials and Bifidobacterium longum infantis subspecies NKU FB3-14; the raw materials are composed of the following components by mass: 30 parts of Cistanche deserticola, 15 parts of ginseng, 10 parts of raspberry, 10 parts of cinnamon, 10 parts of Poria cocos, 10 parts of wolfberry, 10 parts of Chinese yam, 10 parts of Polygonatum sibiricum, 10 parts of Eucommia ulmoides male flowers and 10 parts of oyster; the concentration of Bifidobacterium longum infantis subspecies NKU FB3-14 in the Bifidobacterium longum infantis subspecies NKU FB3-14 composition is 1×10 7 ~1×10 9 CFU / mL; the preservation number of the Bifidobacterium longum subspecies infantis NKUFB3-14 is CGMCC No.25762.

2. The method for preparing the fermentation product of Bifidobacterium longum subspecies infantis NKU FB3-14 according to claim 1, wherein The steps include: The raw materials and water are mixed and then soaked and decocted in sequence to obtain a water extract; Concentrating the aqueous extract to a crude drug amount of 1 g / mL to obtain an extract; Alternatively, the raw material is crushed and mixed with water until the crude drug amount is 1 g / mL to obtain a mixture; Bifidobacterium longum subsp. infantis NKU FB3-14 is inoculated into the extract or the mixture for fermentation to obtain the Bifidobacterium longum subsp. infantis NKU FB3-14 fermentation product.

3. The preparation method according to claim 2, characterized in that The OD of the Bifidobacterium longum subsp. infantis NKU FB3-14 600 The value is 2; the inoculation amount of the Bifidobacterium longum subspecies infantis NKU FB3-14 is 9% to 11% of the volume of the extract.

4. The preparation method according to claim 2, characterized in that The fermentation time is 10-24 hours and the temperature is 36-38°C.

5. Use of the fermented product of Bifidobacterium longum subsp. infantis NKU FB3-14 according to claim 1 or the fermented product of Bifidobacterium longum subsp. infantis NKU FB3-14 obtained by the preparation method according to any one of claims 2 to 4 in preparing food, wherein the food is a food for relieving physical fatigue.

6. Use of the fermented product of Bifidobacterium longum subsp. infantis NKU FB3-14 according to claim 1 or the fermented product of Bifidobacterium longum subsp. infantis NKU FB3-14 obtained by the preparation method according to any one of claims 2 to 4 in the preparation of a product having one or more of the following functions, wherein the product is a non-food product: 1) Improve body muscle tissue damage; 2) Improve exercise capacity; 3) Improve muscle energy metabolism; 4) Improve body endurance; 5) Improve chronic kidney disease; 6) Improve fatigue caused by reproductive factors.

7. The use according to claim 6, characterized in that The improvement of chronic kidney disease includes improving kidney tissue damage and / or reducing the level of kidney tissue damage markers; the kidney tissue damage markers include blood creatinine; the improvement of fatigue caused by reproductive factors includes increasing the testosterone level in testicular cells, reducing the level of oxidative stress in testicular cells, improving testicular tissue damage, improving androgen levels and increasing fertilization rate. One or more of the following.

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