Use of intestinal bacteria that co-produce d-lactate and metabolite thereof d-lactate in predicting exercise capacity

Abstract

A use of intestinal bacteria that co-produce D-lactate and a metabolite thereof D-lactate in predicting exercise capacity. The intestinal bacteria that co-produce D-lactate and the metabolite thereof D-lactate affect the levels of the exercise-related hormones dopamine and adrenaline, thereby significantly impairing exercise capacity. The intestinal bacteria that co-produce D-lactate and the metabolite thereof D-lactate are markers that can be used for routine exercise monitoring and for regulating exercise tolerance.

Description

Application of gut-derived D-lactic acid bacteria and their metabolite D-lactic acid in predicting motor ability Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of gut-co-producing D-type lactic acid bacteria and their metabolite D-type lactic acid in predicting motor ability. Background Technology

[0002] Exercise capacity (EC) refers to a person's ability to participate in sports and training. It is a comprehensive manifestation of factors such as physical form, physical qualities, functions, skills, and psychological abilities. From a biochemical perspective, the level of exercise capacity mainly depends on the ability to supply and utilize energy during exercise. Poor exercise capacity can manifest as non-pathological / physiological fatigue, which can be further caused by central and peripheral mechanisms. These two mechanisms play important roles in the physiological effects of exercise processes, types, intensity, and duration. In prolonged endurance exercise, the energy resources required by muscles are insufficient to maintain and generate the same level of force, leading to fatigue and reduced athletic performance. Studies have shown a positive two-way relationship between exercise and gut microbiota: exercise influences the diversity and species composition of gut microbiota, reshaping the body's gut microbiota structure. Conversely, gut microbiota can also mediate the beneficial effects of exercise on the body and play a very important role in improving athletic performance.

[0003] Lactic acid has long been considered a product of anaerobic metabolism and is often seen as a major culprit in decreased athletic performance, exercise fatigue, and muscle soreness. Lactic acid mainly includes L-lactic acid and D-lactic acid, their main difference being their optical activity. In the human body, L-lactic acid is the predominant form of lactic acid, with a higher content than D-lactic acid. L-lactic acid is primarily produced by the anaerobic glycolysis of pyruvate and is the main component of lactic acid in the body. D-lactic acid, on the other hand, has relatively fewer sources, mainly from fermentation by gastrointestinal bacteria and exogenous intake. Therefore, in most previous studies on lactic acid, L-lactic acid has been the primary focus. L-lactic acid is widely considered an important intermediate product of glucose metabolism during exercise. Recent findings suggest that it is not only not the main cause of exercise fatigue but may even contribute to improved athletic performance to some extent. For example, L-lactic acid is considered a link between glycolysis and the aerobic pathway. It can move intracellularly and intercellularly, maintaining energy homeostasis, playing a signal transduction role, and regulating exercise metabolism. The accumulation of L-lactic acid may, to some extent, inhibit skeletal muscle fatigue caused by increased extracellular potassium ion concentration, thereby improving exercise endurance. Furthermore, studies have shown that during exercise of varying intensities, L-lactic acid can promote the body's utilization of glucose and inhibit fatty acid utilization, thus increasing the body's energy efficiency. These results indicate that L-lactic acid has broad biological activities in the body, including participation in energy metabolism, regulation of immune responses, and the nervous system. In contrast, D-lactic acid has relatively low biological activity in the human body, and its physiological effects have been poorly understood. Because D-lactic acid is present in low amounts in the human body, and its metabolic pathways and physiological effects mainly depend on the human symbiotic microbiota, which differs from that of L-lactic acid, no research has yet been found on whether D-lactic acid directly affects exercise capacity.

[0004] Summary of the Invention

[0005] The purpose of this invention is to provide the application of gut-derived D-type lactic acid bacteria and their metabolite D-type lactic acid in predicting athletic ability. The abundance of D-type lactic acid bacteria is significantly negatively correlated with athletic performance, and D-type lactic acid bacteria and their metabolite D-type lactic acid are used as markers to predict athletic ability.

[0006] To achieve the above objectives, this invention provides an application of gut-derived D-type lactic acid bacteria and their metabolite D-type lactic acid in predicting athletic performance. The abundance of D-type lactic acid bacteria is negatively correlated with athletic performance, and the levels of D-type lactic acid bacteria and D-type lactic acid are negatively correlated with endogenous exercise-related hormones.

[0007] As a preferred embodiment, the D-type lactic acid producing bacteria are intestinal bacteria that can produce D-type lactic acid. Most of these bacteria belong to the lactic acid bacteria class, including Lactobacillus delbrueckii, Lactobacillus bulgaricus, and Lactobacillus acidophilus. In addition to Lactobacillus, we also observed that the intestinal symbiotic Klebsiella pneumoniae can also produce D-type lactic acid, and its abundance is negatively correlated with motor performance.

[0008] Our study found that the gut microbiota of athletes is completely different from that of sedentary individuals, showing a significant decrease in the abundance of D-type lactic acid bacteria. Predictive models suggest a significant negative correlation between the abundance of D-type lactic acid bacteria and athletic performance. By orally supplementing with D-type lactic acid bacteria to predict and intervene in athletic performance, we found that mice supplemented with D-type lactic acid bacteria had significantly lower athletic ability compared to the control group. Oral intervention with the metabolite D-lactic acid of D-type lactic acid bacteria also significantly reduced athletic ability in mice.

[0009] The advantages of this invention are that it provides information on how gut-producing D-type lactic acid bacteria and their metabolite D-lactic acid affect the levels of exercise-related hormones dopamine and adrenaline, thereby significantly impairing athletic performance. D-type lactic acid bacteria and their metabolite D-lactic acid can be used as biomarkers for routine exercise monitoring and for regulating exercise endurance. Furthermore, foods targeting D-type lactic acid bacteria and their metabolite D-lactic acid can be produced without impairing athletic performance. Attached Figure Description

[0010] Figure 1. Abundance of gut symbiotic lactic acid-producing bacteria is significantly negatively correlated with motility; (a) Random forest analysis of the top 20 genera of AT and SS, (b) Relative abundance of the top 10 genera of AT and SS, (c) ROC curves assessing the discriminative accuracy of Lactobacillus and Klebsiella abundance for human cohort endurance exercise performance.

[0011] Figure 2. Oral supplementation with lactic acid-producing bacteria significantly impairs motor function.

[0012] Figure 3. Oral supplementation with inactivated lactic acid-producing bacteria has no significant effect on athletic performance.

[0013] Figure 4. The abundance of D-type lactic acid in lactic acid bacteria metabolites is negatively correlated with motility.

[0014] Figure 5. Supplementing with D-type lactic acid-producing bacteria instead of L-type lactic acid-producing bacteria can significantly impair athletic performance.

[0015] Figure 6. Supplementation with bacterial metabolite D-lactate significantly inhibits motor function.

[0016] Figure 7. D-type lactate is significantly negatively correlated with the levels of exercise-related hormones. Detailed Implementation

[0017] The present invention will now be described in detail with reference to specific embodiments. It should be understood that the following specific embodiments are only for helping those skilled in the art to understand the present invention, and are not intended to limit the present invention. To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available.

[0018] Example 1. The abundance of gut commensal lactic acid bacteria is significantly negatively correlated with motility.

[0019] Studies have revealed significant differences in gut microbiota structure between athletes and sedentary individuals, with significant segregation of the microbial communities. To further screen for potential regulators of athletic performance, we performed random forest analysis and identified the top ten bacterial genera with the greatest variation between AT and SS (Figure 1a). Exercise alters gut microbiota composition, as evidenced by changes in microbial taxonomic distribution (Figure 1b). Therefore, we used ROC analysis to identify gut bacteria that could significantly predict athletic performance. As shown in the figure, the top three genera with the best predictive power were Lactobacillus (AUC = 0.955, P = 0.017) and Klebsiella pneumoniae (AUC = 0.818, P = 0.002) (Figure 1c).

[0020] Example 2. Oral probiotic supplements

[0021] Next, to further understand the roles of lactic acid bacteria and Klebsiella pneumoniae in athletic performance, we conducted an oral supplementation experiment in a mouse model. The study found that mice orally supplemented with lactic acid bacteria and Klebsiella pneumoniae showed a significant decrease in athletic performance in both the roller test and the treadmill test.

[0022] Figure 2 illustrates the impaired motor performance of mice treated with oral LacX (lactic acid bacteria) or Klebsiella pneumoniae. Figure 2a shows a schematic diagram of the mouse model intervention. Figures 2b-d show mice treated with live bacteria (Escherichia coli, LacX, and Klebsiella pneumoniae, 1*10⁹ CFU / d) and tracked using a treadmill running model to monitor distance (Figure 2b), activity time (Figure 2c), and average speed (Figure 2d) over 24 hours. Figure 2e shows the average hourly distance of voluntary wheel activity in mice treated with live bacteria, and the quantification of distance (Figure 2f), time (Figure 2g), and speed (Figure 2h) of mice treated with oral LacX and Klebsiella pneumoniae on a treadmill during fatigue. Figure 2i shows the Kaplan-Meier plot of time spent on a treadmill in mice treated with oral LacX and Klebsiella pneumoniae. The statistical significance of differences between groups was analyzed using a two-tailed Student's t-test.

[0023] Example 3. Oral supplementation with live and heat-killed (HK) D-type lactic acid bacteria

[0024] To further verify whether D-type lactic acid bacteria affect motor performance through their metabolites, we conducted oral supplementation experiments with both live and heat-killed (HK) forms of the aforementioned lactic acid bacteria in a mouse model. The study found that oral supplementation with inactivated D-type lactic acid bacteria (HK LacX) did not have a detrimental effect on motor performance in mice.

[0025] Figure 3 shows that oral administration of HK LacX (high-temperature inactivated lactic acid bacteria) did not impair the motor performance of mice. Figures 3a-c show the treadmill performance of mice after treatment with live LacX or inactivated HK LacX (1*10⁹ cfu / d). The quantification of the speed (Figure 3a), time (Figure 3b), and distance (Figure 3c) of fatigue in mice treated with oral LacX and HK LacX on the treadmill. Figure 3d shows the Kaplan-Meier plot of the time spent on the treadmill by mice treated with oral LacX and HK LacX. The statistical significance of differences between groups was analyzed using a two-tailed Student's t-test.

[0026] Example 4. Comparison of lactate metabolism pathways in sedentary individuals and athletes

[0027] To further determine which active components are enriched by lactic acid-producing bacteria to influence athletic performance, we used gut microbiota metabolic function prediction and found that the lactate metabolism pathway was significantly more active in sedentary individuals than in athletes. Given that lactate is mainly divided into D-lactate and L-lactate, our ELISA analysis revealed that the accumulation of D-lactate in serum and feces by lactic acid-producing bacteria primarily caused the accumulation of D-lactate, while L-lactate was not significantly affected.

[0028] Figure 4 suggests that D-lactic acid, rather than L-lactic acid, is associated with the abundance of LacX and Klebsiella pneumoniae. Figure 4a is a heatmap of lactate metabolism in fecal samples based on PICRUSt analysis. Concentrations of D-lactic acid (Figure 4b) and L-lactic acid (Figure 4c) in serum and feces of wheel-running mice treated with live bacteria (Escherichia coli, LacX, and Klebsiella pneumoniae, 1*10⁹ cfu / d) (n=5 per group). Concentrations of D-lactic acid (Figure 4d) and L-lactic acid (Figure 4e) in serum and feces of endurance treadmill mice treated with PBS, Escherichia coli, LacX, and Klebsiella pneumoniae (n=8).

[0029] Example 5. The role of different types of lactic acid bacteria in athletic performance

[0030] Based on the above findings, we further investigated the role of bacteria producing different types of lactic acid in athletic performance. We administered L-lactate-producing *Bifidobacterium breve* (B. breve) and D-lactate-producing *Lactobacillus delbrueckii* subsp. bulgaricus (L. bulgaricus) orally to mice and then measured their effects on athletic performance. The study found that oral supplementation with D-lactate-producing *L. bulgaricus*, rather than L-lactate-producing *B. breve*, significantly impaired athletic endurance.

[0031] Figure 5 shows that D-lactate-producing *L. bulgaricus*, rather than L-lactate-producing *B. breve*, impairs exercise endurance. Figures 5a and 5b show the D / L lactate levels of cultured *L. bulgaricus*, *B. breve*, and *E. coli* DH5α in vitro. Figures 5c-e show the treadmill performance of mice after treatment with live *L. bulgaricus* or *B. breve* (1*10⁹ CFU / d). The distance (Figure 5c), speed (Figure 5d), and time (Figure 5e) of fatigue in mice treated with oral *L. bulgaricus* and *B. breve* are quantified. Figure 5f shows the Kaplan-Meier plot of treadmill time in mice treated with oral LacX and HK LacX. The statistical significance of differences between groups was analyzed using a two-tailed Student's t-test.

[0032] Example 6. Oral supplementation of D-lactic acid in a mouse model

[0033] Furthermore, to understand the role of D-lactic acid, a metabolite of D-type lactic acid bacteria, in athletic performance, we conducted an experiment in a mouse model involving oral supplementation with D-lactic acid. The study found that mice orally supplemented with D-lactic acid instead of L-lactic acid showed a significant decline in athletic performance in both the roller test and the treadmill test.

[0034] Figure 6. This suggests that D-lactic acid, rather than L-lactic acid, impairs motor performance. Figure 6a: Schematic diagram of the mouse model intervention. Mice (n=5) were treated with PBS, D-lactic acid (20 ng / 200 μL / day), and D-lactic acid (20 ng / 200 μL / day), and a wheel running model was used to monitor 24-hour activity time (Figure 6b) and distance (Figure 6c). Figure 6d: Average hourly distance of voluntary wheel activity in D / L-lactic acid treated mice (n=5). Quantification of distance (Figure 6e), time (Figure 6f), and speed (Figure 6g) of L-lactic acid and D-lactic acid treated mice on the treadmill. Figure 6h: Kaplan-Meier plot of fatigue time in mice orally administered L-lactic acid and D-lactic acid.

[0035] Example 7. Correlation between D-lactic acid and levels of endogenous exercise-related hormones

[0036] Finally, we examined the correlation between D-lactic acid and the level of endogenous exercise-related hormones. The results showed that D-lactic acid bacteria and the level of their metabolite D-lactic acid were positively correlated, while D-lactic acid bacteria and the level of D-lactic acid were significantly negatively correlated with the level of endogenous exercise-related hormones.

[0037] Figure 7 shows the correlation between D-type lactic acid bacteria production and D-type lactic acid and endogenous movement-related hormone levels.

[0038] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of gut-derived D-type lactic acid bacteria and their metabolite D-type lactic acid in predicting motor ability.

2. The application of the gut-derived D-type lactic acid bacteria and their metabolite D-type lactic acid according to claim 1 in predicting motor ability, characterized in that, The D-type lactic acid producing bacteria are Lactobacillus delbrueckii, Lactobacillus bulgaricus, Lactobacillus acidophilus, and Klebsiella pneumoniae, which are intestinal commensal bacteria.

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