Application of lactobacillus plantarum functional metabolite L-carnosine in relieving glucose and lipid metabolism disorder induced by high-starch feed

Abstract

The invention discloses an application of a lactobacillus plantarum functional metabolite L-carnosine in relieving glucose and lipid metabolism disorder induced by a high-starch feed. According to the invention, the L-carnosine is added into the high-starch daily ration according to the proportion and is used for feeding a tested object (such as zebra fish), so that liver fatty degeneration and glycolipid metabolism disorder caused by excessive starch intake can be effectively relieved. The technical scheme provided by the invention can be applied to functional feed development of aquatic animals, and is used for preventing and relieving metabolic abnormalities of cultured fishes caused by high vegetable feed; the food can be further applied to human nutrition intervention, for example, the food can be used as a functional food or a dietary supplement to assist in controlling the balance of blood sugar and blood fat of high-carbon water diet people.

Description

Technical Field

[0001] This invention belongs to the fields of animal husbandry and aquaculture, specifically relating to the application of L-carnosine, a functional metabolite of Lactobacillus plantarum, in alleviating glucose and lipid metabolism disorders induced by high-starch feed. Background Technology

[0002] Currently, obesity and its associated metabolic disorders have become a serious challenge to global public health. Obesity often manifests as a series of pathological states, including insulin resistance, type II diabetes, hyperlipidemia, and fatty liver disease. Excessive carbohydrate intake (such as high-starch, high-sugar diets) is considered a major contributing factor to energy imbalance and obesity, leading not only to body fat accumulation but also to glucose and lipid metabolism disorders by altering the gut microbiome and impairing intestinal barrier function. Existing drug interventions (such as hypoglycemic agents and lipid-lowering drugs) can control obesity-related metabolic abnormalities to some extent, but they are often accompanied by various side effects, limiting their long-term use. Therefore, there is an urgent need to develop safer and more effective intervention strategies to prevent and treat metabolic problems caused by an unhealthy diet.

[0003] In recent years, probiotics and their metabolites have received widespread attention for improving host metabolic health. Previous studies have shown that certain probiotic strains can alleviate obesity symptoms by regulating host energy metabolism. For example, our previous research found that *Lactobacillus plantarum* (GDMCC 1.140), as a probiotic additive, can enhance lipid catabolism in fish under high-starch diet conditions and reduce hepatic fat deposition. However, the specific molecular mechanisms by which probiotics exert their effects, especially how their functional metabolites affect host glucose and lipid metabolism, are not yet fully understood. Through metabolite analysis, we identified that *Lactobacillus plantarum* culture produces a key metabolite—carnosine. L-carnosine is a dipeptide composed of β-alanine and histidine, naturally occurring in the body, and is known to play an important role in regulating the muscle homeostasis and antioxidant defense. L-carnosine and its enantiomer, D-carnosine, have similar biological activities. Based on its potential metabolic regulatory function, carnosine, as a small molecule derived from microorganisms, holds promise as a novel functional factor for intervening in diet-induced metabolic disorders.

[0004] However, to date, no technology has been developed to use L-carnosine to alleviate glucose and lipid metabolism disorders caused by high carbohydrate intake, and systematic studies on its mechanism of action and effective dosage are lacking. Against this backdrop, this invention aims to provide a method for intervening in metabolic disorders induced by a high-starch diet using L-carnosine, elucidating its molecular mechanism of action, and overcoming the shortcomings of existing technologies in terms of safety and efficacy. Summary of the Invention

[0005] The first objective of this invention is to provide the application of L-carnosine, a functional metabolite of Lactobacillus plantarum, in alleviating glucose and lipid metabolism disorders induced by high-starch diets.

[0006] The main objective of this invention is to provide a safe and efficient metabolic regulation method to alleviate glucose and lipid metabolism disorders and related fatty liver damage induced by high-starch feed (or high-carbohydrate diet). Specifically, this invention aims to utilize L-carnosine, a metabolite derived from the probiotic *Lactobacillus plantarum*, to improve problems such as liver fat accumulation and glucose metabolism disorders caused by high starch intake, thereby providing a new solution for optimizing aquatic animal feed and preventing metabolic syndrome in humans.

[0007] To achieve the above objectives, the present invention provides the application of L-carnosine in the preparation of drugs, functional feeds or human metabolic regulators for alleviating glucose and lipid metabolism disorders.

[0008] Preferably, L-carnosine is used in the preparation of drugs, functional feeds, or human metabolic regulators that alleviate glucose and lipid metabolism disorders induced by high starch or carbohydrates.

[0009] Preferably, the application is achieved by adding an effective dose of L-carnosine to feed or diet. The L-carnosine may be an extract produced by fermentation of Lactobacillus plantarum, or it may be a synthetically produced pure product.

[0010] Preferably, the amount of L-carnosine added is 0.2% to 1% (by weight) of the total weight of the feed or diet.

[0011] Preferably, L-carnosine is used in the preparation of drugs, functional feeds or human metabolic regulators that reduce hepatic lipid deposition, protect liver tissue structure, and improve insulin resistance and lipid metabolism disorders.

[0012] The present invention also provides a drug, functional feed or human metabolic regulator for relieving glucose and lipid metabolism disorders, which contains L-carnosine as an active ingredient.

[0013] By adding L-carnosine to a high-starch diet in the above proportions and feeding it to test subjects (such as zebrafish), hepatic steatosis and glucose-lipid metabolism disorders caused by excessive starch intake can be effectively alleviated. The technical solution of this invention can be applied to the development of functional feeds for aquatic animals to prevent and alleviate metabolic abnormalities in farmed fish caused by high-plant-based feeds; it can also be further applied to nutritional intervention in humans, such as as a functional food or dietary supplement to help control blood glucose and lipid balance in people with high-carbohydrate diets.

[0014] Compared with existing technologies, this invention utilizes natural small molecule L-carnosine for metabolic intervention, which has significant advantages and beneficial effects:

[0015] (1) High safety: L-carnosine is a common natural substance in the body and food. It has low toxicity and side effects, and is safer than drug intervention. It can be added to feed or food for a long time.

[0016] (2) Significant intervention effect: Experiments have shown that adding L-carnosine can significantly reduce liver lipid deposition caused by a high starch diet, protect liver tissue structure, and improve symptoms such as insulin resistance and lipid metabolism disorders.

[0017] (3) Clear mechanism: Through transcriptomic analysis, this invention elucidates the mechanism of action of L-carnosine, including the activation of key metabolic pathways such as glycolysis / gluconeogenesis, fatty acid breakdown and peroxisome proliferator-activated receptor (PPAR) signaling, and the inhibition of adverse metabolic changes, demonstrating its efficacy at the molecular level.

[0018] (4) Wide range of applications: The solution of the present invention can be applied to the field of aquaculture to improve the tolerance of fish to high plant-based feed and reduce economic losses; at the same time, it can be derived for human dietary supplementation as a new strategy for intervention of metabolic syndrome, which has important industrialization value. Attached Figure Description

[0019] Figure 1 Images of zebrafish liver tissue stained with H&E. A represents the normal starch diet group (NSF), B the high starch diet group (HSF), C the high starch diet group with low-dose L-carnosine (CAL), and D the high starch diet group with high-dose L-carnosine (CAH). In the HSF group, hepatocytes showed enlargement, degeneration, increased vacuoles (arrows), and disordered structure; while in groups C and D with added L-carnosine, the hepatocyte arrangement returned to a tighter state, and vacuoles were significantly reduced, indicating that L-carnosine has an ameliorative effect on liver tissue lesions.

[0020] Figure 2 Images of zebrafish liver stained with oil red O. AD classification is the same for each group. Figure 1 In the HSF group, a large amount of lipid accumulation was observed in the liver tissue, appearing as red staining areas (indicated by arrows), while the NSF-normal group showed almost no lipid deposition. In the L-carnosine-treated CAL and CAH groups, only sporadic small lipid droplets were observed, indicating that L-carnosine significantly reduced high-starch diet-induced liver fat accumulation.

[0021] Figure 3 The number of differentially expressed genes (DEGs) between each experimental group. The numbers at the top of the red (upregulated) and blue (downregulated) bars represent the number of differentially expressed genes that are upregulated and downregulated, respectively.

[0022] Figure 4Heatmap analysis of differentially expressed genes in zebrafish liver. The horizontal axis represents the experimental groups (NSF, HSF, CAL, CAH), and the vertical axis represents some significantly differentially expressed genes. The color from blue (low expression) to red (high expression) indicates the relative expression level of genes in different groups. It can be seen that a number of metabolism-related genes were upregulated in the HSF group (shown in red in the figure), while the expression levels of these genes were generally decreased in the CAL and CAH groups treated with L-carnosine (the color turned blue), approaching the normal control level. Simultaneously, the expression of some genes suppressed in the HSF group was restored in the L-carnosine treatment group. This indicates that L-carnosine can reverse the abnormal gene expression patterns induced by high-starch diets and reactivate molecular pathways beneficial to glucose and lipid metabolism balance, explaining its mechanism of action in alleviating metabolic disorders at the gene expression level.

[0023] Figure 5 Representative GO enrichment analysis results for upregulated genes (A) and downregulated genes (B). The size and color of the dots in the bubble chart represent the number of related genes and the significance of the entries, respectively. Entries related to lipid metabolism are highlighted with red rectangles.

[0024] Figure 6 Representative KEGG enrichment analysis results for upregulated genes (A) and downregulated genes (B). The size and color of the dots in the bubble chart represent the number of related genes and the significance of the pathways, respectively. Pathways related to carbohydrate-lipid metabolism are highlighted with red rectangles.

[0025] Figure 7 Network diagram of L-carnosine's effects on carbohydrate-lipid metabolism-related pathways and genes. The hexagons and circles in the diagram represent enriched pathways and differentially expressed genes (DEGs) between the CAH and HSF groups, respectively. The size and color of the circles represent the gene's log2 Foldchange value and padj value, respectively. Detailed Implementation

[0026] The specific embodiments of the present invention will be further described in detail below with reference to examples. However, those skilled in the art should understand that these embodiments are only for illustrating the present invention and not for limiting the scope of the present invention.

[0027] Example: An experiment on the effect of L-carnosine on alleviating glucose and lipid metabolism disorders induced by a high-starch diet in zebrafish.

[0028] 1. Experimental materials and model establishment:

[0029] Several healthy adult zebrafish (Danio rerio) were selected and randomly divided into four groups (three replicates per group, n = 20): a normal starch diet control group (NSF group, 20% starch), a high starch diet model group (HSF group, 40% starch), a high starch diet + low-dose L-carnosine group (0.2% by mass, added to the diet, CAL group), and a high starch diet + high-dose L-carnosine group (1% by mass, added to the diet, CAH group). All experimental diets were uniformly mixed and granulated to ensure the stability of nutrient composition and L-carnosine addition.

[0030] Zebrafish were housed in a standard aquarium environment with a water temperature of approximately 28°C and a light-dark cycle of 14 h:10 h. Each group of fish was fed a corresponding diet, and to ensure uniform intake, they were fed twice daily at fixed times (9:00 AM and 5:00 PM) with a fixed amount (3% of total body weight) for 10 weeks. Throughout the experiment, the fish's growth and health status were regularly observed and recorded.

[0031] 2. Growth index measurement:

[0032] After 10 weeks of experimentation, the growth indicators of zebrafish in each group were statistically analyzed. As shown in Table 1, the average weight of zebrafish in the high-starch diet group (HSF group) was significantly higher than that in the normal diet group (NSF group). The weight of fish in the CAL and CAH groups, which received L-carnosine supplementation, was also significantly higher than that in the NSF group, but the difference was not significant compared to the HSF group. There was no statistically significant difference in body length among the groups. This indicates that the high-starch diet successfully induced an obesity model (weight gain) in zebrafish, while the addition of L-carnosine did not significantly affect normal growth and development.

[0033] Table 1 Comparison of growth indicators (body weight, body length) of zebrafish in different groups

[0034] Note: Values ​​are mean ± standard error. * indicates P < 0.05.

[0035] 3. Liver histological analysis:

[0036] Liver tissues were collected from each group of zebrafish and sectioned in paraffin. Morphological changes were assessed using hematoxylin and eosin (H&E) staining. Figure 1In the normal diet group (NSF), hepatocytes showed regular morphology, tight cell connections, clear nuclear membranes, and homogeneous chromatin. In the high-starch diet group (HSF), hepatocytes exhibited significant swelling, with cytoplasm filled with vacuoles of varying sizes, nuclei compressed to the periphery, and disordered normal hepatic plate structure. These pathological changes suggest severe fatty liver degeneration and damage in the HSF group. In contrast, the liver tissue damage in the two groups supplemented with L-carnosine was significantly reduced: hepatocyte structure was more intact, the degree of vacuolar degeneration was greatly reduced, and the position of the nuclei tended to be normal. Histological quantitative analysis (such as hepatocyte vacuolation score) also showed that the liver damage scores of the CAL and CAH groups were significantly lower than those of the HSF group. Therefore, L-carnosine intervention effectively improved the pathological damage to zebrafish liver tissue caused by high starch intake.

[0037] Hepatic lipid accumulation was further assessed using Oil Red O staining. The results showed ( Figure 2 In the HSF group, large areas of red staining were observed in liver sections, indicating significant lipid accumulation. In contrast, the NSF normal group showed only scattered lipid droplets, and the CAL and CAH groups also showed only a small number of small lipid droplets. This demonstrates that high starch intake leads to severe fat deposition in fish livers, while the addition of L-carnosine can significantly reduce lipid accumulation. Combined with H&E and Oil Red O results, it can be confirmed that L-carnosine has a significant inhibitory and reversal effect on high-starch diet-induced hepatic steatosis, protecting the normal structure and function of liver tissue.

[0038] 4. Molecular biological analysis:

[0039] To elucidate the molecular mechanism by which L-carnosine improves glucose and lipid metabolism disorders, this invention performed transcriptomic sequencing analysis on the livers of zebrafish from different groups. Gene expression profiles in the livers of different groups were compared using RNA-Seq to screen for differentially expressed genes (DEGs). The results showed that ( Figure 3 In the high-starch diet group (HSF vs. NSF), the expression of hundreds of genes was significantly altered, with approximately 215 genes upregulated and 399 genes downregulated, reflecting the broad impact of high starch intake on fish liver metabolism and function. In contrast, the addition of L-carnosine corrected the abnormal expression of many genes induced by high starch. Figure 4 Compared to the HSF group, the low-dose group (CAL) showed a large number of upregulated genes (compensatory upregulation relative to the HSF group), while the high-dose group (CAH) showed the most gene downregulation changes. Notably, the CAL group had the fewest differentially expressed genes compared to the CAH group, indicating that the 0.2% and 1.0% doses of L-carnosine had similar overall gene regulatory effects, and the high dose did not cause excessive or abnormal expression changes.

[0040] Gene Ontology (GO) enrichment analysis of differentially expressed genes revealed ( Figure 5In the HSF group, upregulated genes were significantly enriched in biological processes such as lipid responses, consistent with lipid metabolism disorders induced by high starch intake. In the CAL and CAH groups, the upregulated gene sets compared to the HSF group involved multiple metabolic-related processes, including triglyceride homeostasis, lipid homeostasis, immune response, and motor function. The enrichment of triglyceride and lipid homeostasis-related processes, in particular, indicates that dietary L-carnosine supplementation can activate the body's lipid metabolism regulation mechanisms, helping to restore lipid metabolism imbalances caused by high-starch diets. On the other hand, downregulated genes in the HSF group were enriched in pathways such as glucose and vitamin metabolism, showing that high starch intake inhibits normal glucose metabolism pathways; while the L-carnosine-treated group showed downregulated enrichment of some genes related to cell cycle regulation and DNA repair. These results suggest that L-carnosine, on the one hand, corrects the metabolic pathway inhibition caused by high starch, and on the other hand, may also exert a protective effect by inhibiting abnormal cell proliferation signals.

[0041] Further KEGG metabolic pathway enrichment analysis revealed a key pathway by which L-carnosine regulates glucose and lipid metabolism. Figure 6 Compared to the HSF group, the differentially regulated genes in the CAL and CAH groups were significantly enriched in multiple carbohydrate and lipid metabolism pathways, such as the pentose and uronic acid exchange pathway and fatty acid catabolism pathway. Especially in the high-dose groups (CAH vs. HSF), the upregulated genes were also enriched in glycolysis / gluconeogenesis, pyruvate metabolism, PPAR signaling, and unsaturated fatty acid biosynthesis pathways. These pathways are closely related to the balance of glucose utilization, fatty acid oxidation, and lipid synthesis in vivo. The upregulated pathways specific to the high-dose L-carnosine group also included amino acid metabolism pathways such as tryptophan, tyrosine, and β-alanine metabolism. β-alanine is a component of carnosine, suggesting that additional L-carnosine may influence its own metabolic pathways through feedback. These molecular-level changes are highly consistent with phenotypic observations: the CAH group had significantly less fat accumulation in the liver than the CAL group, corresponding to its more significant role in promoting fatty acid catabolism and utilization.

[0042] Through specific analysis of differentially expressed genes, this invention further identified several key metabolic genes regulated by L-carnosine. Figure 7For example, in glycolysis / gluconeogenesis, genes such as phosphoglycerate mutase 2 (pgam2), fructose-1,2-bisphosphatase 1b (fbp1b), and phosphoenolpyruvate carboxykinase b (pckb) were significantly upregulated in the CAH group. Regarding fatty acid metabolism, stearoyl-CoA desaturase (scd), long-chain fatty acid elongase 6 (elovl6), and 8b (elovl8b), which are involved in the synthesis of unsaturated fatty acids, showed increased expression after the addition of L-carnosine. Genes mediating lipid transport and storage balance, such as diacylglycerol acyltransferase 2 (dgat2), cholesterol efflux transporter G5 (abcg5), and apolipoprotein A4-like protein 1 (apoa4b.1), were also upregulated. These gene changes collectively promoted the breakdown and utilization of fatty acids and lipid export in the liver, reducing abnormal lipid accumulation within the liver. These molecular mechanisms clearly explain why L-carnosine can effectively alleviate glucose and lipid metabolism disorders induced by high-starch diets: by reprogramming the liver's transcriptional regulatory network, L-carnosine enhances the body's ability to process excess carbohydrates and lipids, thus restoring the damaged metabolic balance.

[0043] Based on the results of the above embodiments, it can be confirmed that the technical solution provided by the present invention is feasible and effective. By adding L-carnosine to a high-starch feed, regardless of whether the dose is low or high, it can protect the liver and improve glucose and lipid metabolism in a zebrafish model. This discovery lays an experimental foundation for the development of novel functional feed additives and human nutritional intervention formulations. Related mechanism studies show that L-carnosine achieves its function by regulating metabolic pathways through multiple targets, and has good application prospects. Of course, those skilled in the art, after reading this specification, can make various changes and improvements based on the concept of the present invention, such as applying L-carnosine to other animal models or clinical trials to verify its similar efficacy in mammals (including humans). These equivalent variations based on the spirit of the present invention should all fall within the scope of protection claimed by the present invention.

Claims

1. Use of L-carnosine in the preparation of a medicament, functional feed or human metabolic regulator for relieving glycolipid metabolism disorder.

2. Use according to claim 1, characterized in that, Use of L-carnosine in the preparation of a medicament, functional feed or human metabolic regulator for relieving high starch or carbohydrate-induced glycolipid metabolism disorder.

3. Use according to claim 1, characterized in that, The use is achieved by adding an effective amount of L-carnosine to feed or diet.

4. Use according to claim 3, characterized in that, The L-carnosine is an extract produced by L. plantarum fermentation or a pure synthetic product.

5. Use according to claim 3, characterized in that, The amount of L-carnosine added is 0.2% to 1% by mass of the total weight of the feed or diet.

6. Use according to claim 1, characterized in that, Use of L-carnosine in the preparation of a medicament, functional feed or human metabolic regulator for reducing liver lipid deposition, protecting liver tissue structure, improving insulin resistance and lipid metabolism disorder.

7. A medicament, a functional feed or a human metabolic regulator for alleviating disorders of glycolipid metabolism, characterized by, It contains L-carnosine as an active ingredient.

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