Bifidobacterium longum and longan aril composition capable of synergistically relieving chronic fatigue

Abstract

The invention provides a bifidobacterium longum and longan aril composition capable of synergistically relieving chronic fatigue, the bifidobacterium longum is named as BB36, the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO.34123, and the preservation date is April 8, 2025. By improving the levels of stress hormones and neurotransmitters and reducing the serum inflammation level, the strain significantly improves the chronic fatigue symptom and improves the athletic ability; the invention further develops a bifidobacterium longum and longan aril composition capable of synergistically relieving chronic fatigue, and through compounding of probiotics and longan aril extract, it is found that the probiotics and the longan aril extract can cooperate with each other, and the composition has a synergistic effect in relieving chronic fatigue.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to a combination of Bifidobacterium longum and longan pulp that synergistically relieves chronic fatigue. Background Technology

[0002] Chronic fatigue syndrome (CFS) is a chronic, disabling fatigue disorder in which patients experience fatigue even with daily activities and simple work tasks, and this fatigue cannot be relieved by rest. Current research suggests that weakened immunity, excessive stress, sleep disorders, and insufficient energy are potential causes of CFS, and there is an urgent need for safe and effective intervention programs in clinical practice.

[0003] Longan pulp, the aril of the longan plant (Dimocarpus longan Lour.), is a traditional food and medicine ingredient with the effects of nourishing the heart and spleen, replenishing blood and calming the mind, and improving intelligence. Probiotics, on the other hand, are gradually gaining attention due to their beneficial properties such as enhancing immunity, regulating gut microbiota balance, and alleviating mood-related discomfort.

[0004] However, existing technologies for treating chronic fatigue often have limitations: when longan pulp is used alone, its effect on regulating hormone homeostasis is limited, making it difficult to effectively improve CFS-related inflammatory imbalances and peripheral fatigue symptoms; when probiotics are used alone, although they can play a role in immune regulation, they lack the synergistic effect of nutritional support and neurotransmitter regulation, resulting in poor overall fatigue relief.

[0005] Therefore, given the limited effectiveness of existing single intervention methods and the lack of synergistic enhancement strategies, screening probiotic strains with clear efficacy in relieving chronic fatigue, exploring their synergistic mechanism with longan pulp extract, and developing a composition that can improve chronic fatigue symptoms in multiple targets and in all aspects has become an urgent technical problem to be solved in this field. Summary of the Invention

[0006] In view of the problems existing in the prior art, the purpose of this invention is to provide probiotics that have good effects in preventing and treating chronic fatigue, and to explore their synergistic therapeutic effects with traditional Chinese medicine ingredients, so as to provide a combination of Bifidobacterium longum and longan pulp that synergistically relieves chronic fatigue.

[0007] Because of the complex composition of substances that are both food and medicine, the mechanisms by which they help improve chronic fatigue symptoms are unclear. This leads to different possible synergistic effects when different probiotics are combined with different extracts, resulting in either antagonistic, synergistic, or additive effects. Therefore, the inventors conducted research to discover food and medicine homologous substances that can achieve synergistic effects when used in combination with Bifidobacterium longum BB36 as described in this application. The food and medicine homologous materials screened in the early stages of the research included longan and wolfberry. However, experiments showed that while both longan pulp extract and wolfberry extract, when used alone, could improve the levels of stress hormones and neurotransmitters, when combined with Bifidobacterium longum BB36, the combination of BB36 and wolfberry extract showed a largely additive synergistic effect in mice with chronic fatigue. In contrast, the combination of longan pulp extract and Bifidobacterium longum BB36 exhibited a significant synergistic effect.

[0008] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a Bifidobacterium longum strain, named BB36, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), classified as Bifidobacterium longum, with accession number CGMCC NO. 34123 and deposit date of April 8, 2025.

[0009] In another aspect, the present invention provides a composition of Bifidobacterium longum and longan pulp that synergistically relieves chronic fatigue, characterized in that the active ingredients of the composition include Bifidobacterium longum and longan pulp extract; The Bifidobacterium longum strain is named BB36. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) and classified as Bifidobacterium longum. Its accession number is CGMCC NO.34123 and its accession date is April 8, 2025. In the composition, the final therapeutic dose of Bifidobacterium longum BB36 is at a viable count of not less than CFU / d / kg body weight.

[0010] Furthermore, the longan pulp extract is a water extract of longan pulp, and the extraction method includes: Take longan pulp, add 8-15 times its weight of water, and heat to extract 2-5 times, each time for 0.5-1.5 hours. Then filter to obtain the extract, combine the extracts obtained from each extraction, concentrate under reduced pressure, and freeze dry to obtain longan pulp extract.

[0011] Furthermore, the final therapeutic dose of the longan pulp extract is 3-9 g / d / kg body weight.

[0012] Furthermore, the dosage form of the composition includes lyophilized powder, capsules, tablets, or granules.

[0013] In another aspect, the present invention provides the application of the above-mentioned Bifidobacterium longum BB36, or a combination of Bifidobacterium longum and longan pulp that synergistically relieves chronic fatigue, in the preparation of a product for preventing and treating chronic fatigue, said product being food or pharmaceutical.

[0014] Furthermore, the drug is one or more of the following: (1) The drug can reduce the concentration of cortisol in serum; (2) The drug can increase the concentration of serotonin in serum; (3) The drug can reduce serum inflammation levels.

[0015] Furthermore, the reduction in serum inflammation levels includes: reducing the concentrations of pro-inflammatory factors IL-6 and TNF-α.

[0016] In another aspect, the present invention provides a probiotic composition comprising the above-mentioned Bifidobacterium longum BB36, or the above-mentioned Bifidobacterium longum and longan pulp composition which synergistically relieve chronic fatigue.

[0017] Information on strain preservation: The strain of *Bifidobacterium longum* was named BB36 and is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its classification name is *Bifidobacterium longum*, accession number is CGMCC NO. 34123, and the deposit date is April 8, 2025.

[0018] The advantages of this invention compared to the prior art are as follows: 1. This application screened and obtained a probiotic strain that can relieve chronic fatigue. The strain was identified as Bifidobacterium longum after preservation and identification, with the preservation number CGMCC NO. 34123 and the classification name Bifidobacterium longum.

[0019] 2. This application found that the use of Bifidobacterium longum BB36 can significantly improve the levels of stress hormones and neurotransmitters (specifically: effectively reduce the concentration of cortisol in serum while increasing the concentration of serotonin), reduce serum inflammation levels, thereby significantly improving chronic fatigue symptoms and enhancing exercise capacity; its effect is superior to that of Bifidobacterium animalis BB12, which has already been developed and marketed.

[0020] 3. The composition of Bifidobacterium longum BB36 and longan pulp water extract described in this application has a better intervention effect on chronic fatigue symptoms than Bifidobacterium animalum BB12, reaching the level of drug intervention. This has important practical significance for the development of highly effective drugs for the prevention and treatment of chronic fatigue. Attached Figure Description

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 The effects of different concentrations of longan pulp extract on hormone levels in mice are shown; where A represents cortisol and B represents serotonin. Figure 2 The effects of the composition on hormone and neurotransmitter levels in mice are shown; wherein, A is cortisol; and B is serotonin. Figure 3 The effect of the composition on inflammation levels in mice is shown; where A is IL-6 and B is TNF-α. Figure 4 The study demonstrates the effect of the composition on improving the motor function of mice; where A represents running time and B represents grip strength. Detailed Implementation

[0022] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0023] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0024] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0025] In the following examples, *Bifidobacterium longum* BB36 was provided by Shaanxi Yiruikang Biotechnology Co., Ltd., and the strain was deposited at the China General Microbiological Culture Collection Center, derived from the human gut. *Bifidobacterium animalis* BB12 was obtained from Chr. Hansen AG, Denmark.

[0026] Example 1 – Preparation for Microbial Culture 1) Preparation of Bifidobacterium longum BB36 cells: A probiotic strain was obtained by isolating human intestinal flora and identified as Bifidobacterium longum. It was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34123 and deposit date of April 8, 2025.

[0027] The isolated *Bifidobacterium longum* BB36 was passaged twice in modified MRS liquid medium, and then inoculated into modified MRS liquid medium at a 2% inoculum. The culture was carried out at 37 ± 1 °C and 0% oxygen for 12 h. After centrifugation (4000 rpm, 10 min) to remove the supernatant, the bacteria were washed twice with PBS. Before gavage, the bacterial suspension was resuspended in 0.9% physiological saline and the concentration was adjusted to [missing value]. CFU / g.

[0028] 2) Preparation of Bifidobacterium animalis BB12 cells: Bifidobacterium animalis BB12 was passaged twice in modified MRS liquid medium and then inoculated at a 2% inoculum. The culture was carried out at 37 ± 1 °C and 0% oxygen for 12 h. After centrifugation (4000 rpm, 10 min) to remove the supernatant, the cells were washed twice with PBS. Before gavage, the cells were resuspended in 0.9% physiological saline and the bacterial suspension concentration was adjusted. CFU / g.

[0029] Example 2 – Preparation of Longan Pulp Extract This embodiment provides a method for extracting water extracts from longan pulp, including the following steps: Take longan pulp, add 10 times its weight of water, and heat to extract three times, one hour each time. Then filter to obtain the extract, combine the extracts from each extraction, concentrate under reduced pressure, and freeze dry to obtain the water extract of longan pulp.

[0030] When administered by gavage, the drug is dissolved in distilled water to prepare doses of 3, 6, or 9 g / kg (raw drug weight) for low, medium, or high doses, once daily at 20 mL / kg BW for 28 days.

[0031] Example 3 – Preparation of Lycium barbarum extract This embodiment provides a method for preparing a water extract of wolfberry, including the following steps: Weigh out wolfberries and soak them in deionized water for 12 hours. Boil them over low heat for 3 hours, filter them with gauze, and then add deionized water to repeat the extraction once. Combine the two extracts, boil and concentrate them to prepare low, medium and high doses of 3, 6 and 9 g / kg. Take once a day at 20 mL / kg BW for 28 days.

[0032] Example 4 – Preparation of Animal Model and Animal Experiments Eight-month-old male BALB / c mice (n=8) were used as experimental animals. The model was established using intense sleep deprivation. One sleep deprivation cycle was 48 hours, followed by a 24-hour rest period. A total of six sleep deprivation cycles were performed.

[0033] The first batch of animal experiments screened the dosage of longan pulp extract, and was divided into 8 groups: control group, model group, high-dose longan pulp extract group, medium-dose group, and low-dose group (9, 6, 3 g / kg), and high-dose, medium-dose, and low-dose wolfberry extract group (9, 6, 3 g / kg), with 8 mice in each group, for a total of 80 mice.

[0034] The second batch of animal experiments involved the synergistic evaluation of a combination of Bifidobacterium longum BB36, Bifidobacterium animalis BB12, longan pulp extract, and wolfberry extract. Eight-month-old male BALB / c mice (n=8) were used to establish the model through intense sleep deprivation, and simultaneously administered longan pulp water extract (6 g / kg) and Bifidobacterium longum BB36 by gavage. CFU / d / kg), Bifidobacterium animalis BB12 ( CFU / d / kg), a mixture of probiotics and longan pulp extract ( CFU / d / kg + 6 g / kg), a mixture of probiotics and wolfberry extract ( CFU / d / kg + 9 g / kg).

[0035] By examining the effects of the intervention on serum cortisol and serotonin concentrations in mice, the optimal dosage of longan pulp for alleviating chronic fatigue syndrome was screened. Figure 1 The results showed that after 28 days of intervention with different doses of longan pulp extract, low-dose (3 g / d / kg), medium-dose (6 g / d / kg), and high-dose (9 g / d / kg) longan pulp extract, as well as low-dose (3 g / d / kg), medium-dose (6 g / d / kg), and high-dose (9 g / d / kg) wolfberry extract all improved cortisol and serotonin levels. The medium-dose (6 g / d / kg) and high-dose (9 g / d / kg) longan pulp extract showed the best intervention effects, with cortisol levels decreasing by 40% and 46%, respectively, compared to the model group. Figure 2 (A); serotonin levels increased by 2.2 times and 2.4 times ( ). Figure 2 (B)

[0036] As shown in Table 1, the combined intervention of *Bifidobacterium longum* BB36 and longan pulp extract (6 g / d / kg) for 28 days significantly improved the symptoms of chronic fatigue in mice. The total distance traveled and the time spent in the central region after intervention with the *Bifidobacterium longum* BB36 and longan pulp extract combination were 1.86 times and 5.05 times that of the model group, respectively, and there was no significant difference in the central region time compared with the control group. Similarly, the combined intervention of *Bifidobacterium animalis* BB12 and longan pulp extract (6 g / d / kg) for 28 days also significantly improved the symptoms of chronic fatigue in mice. Compared with the model group, the total distance traveled and the time spent in the central region were 1.50 times and 3.95 times that of the model group, respectively. This indicates that the combination of *Bifidobacterium longum* BB36 and longan pulp extract (6 g / d / kg) is more effective than the combination of *Bifidobacterium animalis* BB12 and longan pulp extract (6 g / d / kg) in improving chronic fatigue in mice. Compared with the model group, after mice were treated with a mixture of Lycium barbarum extract (9 g / d / kg) and Bifidobacterium longum BB36 and Bifidobacterium animalis BB12 for 28 days, the total distance traveled was 1.36 times and 1.39 times that of the model group, respectively, and the time spent in the central region was 3.73 times and 3.83 times that of the model group, respectively.

[0037]

[0038] like Figure 2 The results showed that after 28 days of co-treatment with *Bifidobacterium longum* BB36 and longan pulp extract (6 g / d / kg), the levels of hormones and neurotransmitters in mice were significantly improved. Compared with the model group, the cortisol level in the *Bifidobacterium longum* BB36 and longan pulp extract (6 g / d / kg) intervention group was reduced by 63%. Figure 2 (A); serotonin levels increased 3.03 times ( ) Figure 2 (B) Intervention in mice with Bifidobacterium animalis BB-12 and longan pulp extract (6 g / d / kg) for 28 days significantly improved the levels of stress hormones and neurotransmitters. Compared with the model group, the cortisol level in the intervention group of Bifidobacterium animalis BB-12 and longan pulp extract (6 g / d / kg) decreased by 36%. Figure 2 (A); serotonin levels increased 2.35 times ( Figure 2 (Middle B). This indicates that the combination of *Bifidobacterium longum* BB36 and longan pulp extract (6 g / d / kg) is more effective than the combination of *Bifidobacterium longum* BB-12 and longan pulp extract (6 g / d / kg) in improving hormone levels and neurotransmitter secretion in mice. Compared with the model group, the cortisol level in mice in the intervention group of *Bifidobacterium longum* BB36 and *Lycium barbarum* extract (9 g / d / kg) was reduced by 46%. Figure 2 (A); serotonin levels increased 2.63 times ( Figure 2In the intervention group (B), the levels of cortisol in mice were reduced by 40% due to the use of Bifidobacterium animalis BB12 and Lycium barbarum extract (6 g / d / kg). Figure 2 (A); serotonin levels increased 2.53 times ( Figure 2 (B)

[0039] like Figure 3 The results showed that co-intervention of Bifidobacterium longum BB36 and longan pulp extract (6 g / d / kg) in mice for 28 days significantly improved serum inflammation levels. Compared with the model group, the levels of IL-6 and TNF-α in the intervention group of Bifidobacterium longum BB36 and longan pulp extract (6 g / d / kg) decreased by 25% and 27%, respectively. Similarly, Bifidobacterium animalis BB-12 and longan pulp extract (6 g / d / kg) also significantly improved serum inflammation levels. Compared with the model group, the levels of IL-6 and TNF-α in the intervention group of Bifidobacterium animalis BB12 and longan pulp extract (6 g / d / kg) decreased by 13% and 18%, respectively. This indicates that the combination of Bifidobacterium longum BB36 and longan pulp extract (6 g / d / kg) is more effective than the combination of Bifidobacterium animalis BB-12 and longan pulp extract (6 g / d / kg) in reducing inflammation levels. Compared with the model group, the levels of IL-6 and TNF-α in mice in the intervention group of Bifidobacterium longum BB36 and Lycium barbarum extract (9 g / d / kg) decreased by 13% and 23%, respectively, while the levels of serum IL-6 and TNF-α in mice in the intervention group of Bifidobacterium animalis BB12 and Lycium barbarum extract (9 g / d / kg) decreased by 16% and 23%, respectively.

[0040] The effects of various substance interventions on peripheral fatigue in chronic fatigue were evaluated by assessing the motor abilities of mice. The assessment of mouse motor abilities primarily included measuring running time and grip strength. For example... Figure 4 As shown, the combination of *Bifidobacterium longum* BB36 and longan pulp extract (6 g / d / kg) significantly improved peripheral fatigue symptoms in mice. The running time and grip strength of mice in the *Bifidobacterium longum* BB36 and longan pulp extract (6 g / d / kg) intervention group were 1.88 times and 1.8 times that of the model group, respectively. The running time and grip strength of mice in the *Bifidobacterium animalis* BB12 and longan pulp extract (6 g / d / kg) intervention group were both 1.6 times that of the model group. This indicates that the combination of *Bifidobacterium longum* BB36 and longan pulp extract (6 g / d / kg) is more effective than the combination of *Bifidobacterium animalis* BB12 and longan pulp extract (6 g / d / kg) in alleviating peripheral fatigue. The running time and grip strength of mice in the intervention groups of Bifidobacterium longum BB36 and Lycium barbarum extract (9 g / d / kg) were 1.55 times and 1.68 times that of the model group, respectively; the running time and grip strength of mice in the intervention groups of Bifidobacterium longum BB12 and Lycium barbarum extract (9 g / d / kg) were 1.54 times and 1.65 times that of the model group, respectively.

[0041] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A type of Bifidobacterium longum, characterized in that, Named BB36, this strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), classified as *Bifidobacterium longum*, with accession number CGMCC NO. 34123 and deposit date of April 8, 2025.

2. A composition of Bifidobacterium longum and longan pulp that synergistically relieves chronic fatigue, characterized in that, The active ingredients of the composition include Bifidobacterium longum and longan pulp extract; The Bifidobacterium longum strain is named BB36. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) and classified as Bifidobacterium longum. Its accession number is CGMCC NO. 34123 and its accession date is April 8, 2025. In the composition, the final therapeutic dose of Bifidobacterium longum BB36 is at a viable count of not less than CFU / d / kg body weight.

3. The composition according to claim 2, characterized in that, The longan pulp extract is a water extract of longan pulp, and the extraction method includes: Take longan pulp, add 8-15 times its weight of water, and heat to extract 2-5 times, each time for 0.5-1.5 hours. Then filter to obtain the extract, combine the extracts obtained from each extraction, concentrate under reduced pressure, and freeze dry to obtain longan pulp extract.

4. The composition according to claim 2, characterized in that, The final therapeutic dose of the longan pulp extract is 3-9 g / d / kg body weight.

5. The composition according to any one of claims 2 to 4, characterized in that, The dosage forms of the composition include lyophilized powder, capsules, tablets, or granules.

6. The use of a Bifidobacterium longum as described in claim 1, or a composition as described in any one of claims 2 to 5, in the preparation of a product for preventing and treating chronic fatigue, wherein the product is a food or a pharmaceutical.

7. The application according to claim 6, characterized in that, The drug is one or more of the following: (1) The drug can reduce the concentration of cortisol in serum; (2) The drug can increase the concentration of serotonin in serum; (3) The drug can reduce serum inflammation levels.

8. The application according to claim 7, characterized in that, The reduction in serum inflammation levels includes: reducing the concentrations of pro-inflammatory factors IL-6 and TNF-α.

9. A probiotic composition, characterized in that, The composition comprises Bifidobacterium longum as described in claim 1, or Bifidobacterium longum as described in any one of claims 2 to 5, and longan pulp.

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