Fermentation composition based on signal peptide induction and application thereof

Abstract

The invention belongs to the technical field of biological medicine and functional food, and discloses a fermentation composition based on signal peptide induction, which is prepared by the following steps: step 1, mixing astragalus membranaceus, lotus leaf, poria cocos and pericarpium citri reticulatae according to the mass ratio of (3-5): (2-4): (2-3): (1-2) to prepare a compound raw material; the fermentation composition is remarkable in effect and multi-target, the food intake can be reduced by reducing mRNA expression of hypothalamus neuropeptide Y and rat-related peptide and increasing the content of glucagon-like peptide-1, lipid metabolism can be regulated and controlled by improving mRNA expression of liver farnesoid X receptors, AMP-dependent protein kinase and carnitine palmitoyl transferase-1, and the food intake can be reduced by increasing the content of glucagon-like peptide-1. The abundance of intestinal bifidobacteria can be improved, and the expression of brown fat heat production related genes is promoted to balance intestinal flora; the safety of the composition is subjected to dual verification of animal and crowd tests, the weight and blood fat indexes of obese rats and obese crowds are remarkably improved, and the liver and kidney functions are not damaged.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine and functional food, and specifically relates to a fermentation composition based on signal peptide induction and application thereof. BACKGROUND

[0002] With the change of global dietary structure and lifestyle, obesity has become a recognized chronic metabolic disease, and is closely related to type 2 diabetes, hypertension, digestive system cancer and other complications.

[0003] Among the existing obesity intervention means, traditional Chinese medicine compound shows significant safety advantages due to its "homology of medicine and food", but it also has some obvious shortcomings: first, the action pathway is relatively single, mainly focusing on the regulation of a single metabolic pathway, and lacking the synergistic effect of multiple targets; second, the efficacy is limited, the release rate of effective components of unfermented traditional Chinese medicine compound is low, and substances such as n-hexyl alcohol and n-hexanol affecting flavor and stability are easily produced; third, the clinical verification is insufficient, most studies only stay at the animal experiment stage, and the sample size of human trials is small and the coverage of the population is limited, which is difficult to support the industrial application of the product; although fermentation technology has been widely used in the optimization of traditional Chinese medicine compound, there are still some shortcomings in the existing technology: the strain screening lacks pertinence, and the special strain cannot be matched by combining the theory of "tonifying qi and invigorating spleen, resolving phlegm and eliminating dampness" in traditional Chinese medicine; high-efficiency inducing factors are not introduced in the fermentation process, resulting in low fermentation efficiency of the microbial community and insufficient generation of anti-obesity active substances, therefore, it is urgent to develop an anti-obesity composition based on traditional Chinese medicine theory, efficiently fermented by special strains, and verified by animals and humans, which is the key to break through the bottleneck of the existing technology. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides a fermentation composition based on signal peptide induction and application thereof to solve the problems raised in the background.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical scheme: a fermentation composition based on signal peptide induction, prepared by the following steps: Step one: mix radix astragali, lotus leaf, poria cocos and dried tangerine or orange peel according to the mass ratio (3-5):(2-4):(2-3):(1-2) to prepare compound raw materials, add 10 times the mass of deionized water for extraction, and obtain compound water extract after filtration; Step two: add 1% stachyose to the above compound water extract, adjust the pH value to 6.5-7.0, inoculate 4.1% of Limosilactobacillus fermentum grx08 bacterial liquid by volume, and add signal peptide with a final concentration of 0.1-0.3 mmol / L; Step three: fermentation for 49.50 hours in an anaerobic environment at 37℃, and then centrifugation to obtain the fermentation composition.

[0006] Preferably, the temperature range of the leaching in step one is 90-100℃, and the leaching time range is 1.5-2 hours.

[0007] Preferably, the bacterial concentration of the Limosilactobacillus fermentum grx08 bacterial solution in step two is 1×10 8 -1×10 9 CFU / mL.

[0008] Preferably, the amino acid sequence of the signal peptide in step two is His-Gly-Glu-Gly-Thr-Phe-Ser-Ser-Glu, in which Gly provides good flexibility of the peptide chain, the polar residues of Glu and Ser can maintain structural stability under a fermentation environment with pH 6.5-7.0, and the hydrophobic fragment of Phe helps signal recognition and transmembrane transmission.

[0009] Preferably, the centrifugation speed in step three is 8000r / min, and the centrifugation time is 15 minutes.

[0010] Preferably, the fermentation composition in step three is detected by UPLC-MS / MS, and the results show that the fermentation composition contains at least 53 potential substances with anti-obesity effects, and the content of astragaloside in the fermentation composition is increased by more than 30% compared with the unfermented compound water extract, the lipase activity inhibition rate of the fermentation composition is ≥60%, and no n-hexyl aldehyde or n-hexyl alcohol is detected.

[0011] An application of a fermentation composition based on signal peptide induction in the preparation of an anti-obesity product.

[0012] Preferably, the anti-obesity product includes an oral liquid, a solid beverage, a pressed tablet candy, or a capsule.

[0013] Preferably, the anti-obesity product is used for reducing body weight, reducing body fat, improving dyslipidemia, or regulating TCM qi deficiency, phlegm-damp, and damp-heat constitutions.

[0014] An application of a signal peptide in inducing Limosilactobacillus fermentum grx08 fermentation of a medicinal and edible compound, which is used to improve fermentation efficiency and the generation amount of anti-obesity active substances.

[0015] Compared with the prior art, the beneficial effects of the present application are as follows: This fermentation composition exhibits significant efficacy and multiple targets. It can reduce food intake by decreasing the expression of hypothalamic neuropeptide Y and muscarinic-related peptide mRNA, and increasing glucagon-like peptide-1 content. It can regulate lipid metabolism by increasing the expression of hepatic farnesoid X receptor, AMP-dependent protein kinase, and carnitine palmitoyltransferase-1 mRNA. It can also increase the abundance of Bifidobacterium spp. in the gut and promote the expression of brown adipose tissue-related genes to balance the gut microbiota. Its safety has been verified through both animal and human trials. It has not only significantly improved the weight and blood lipid levels of obese rats and obese individuals, but also did not cause damage to liver and kidney function. At the same time, it has outstanding industrialization potential, with well-defined fermentation parameters. Signal peptide induction can improve efficiency and the content of active substances. The product is stable and has a good flavor. It can be directly developed into various anti-obesity products such as oral liquids and solid beverages, possessing both scientific value and market prospects. Detailed Implementation

[0016] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Example 1: Preparation of Fermentation Composition A signal peptide-induced fermentation composition is prepared by the following steps: Step 1: Mix 20g of Astragalus membranaceus, 15g of lotus leaf, 10g of Poria cocos, and 9g of dried tangerine peel to prepare compound raw materials. Add 100mL of 10 times the mass of deionized water for extraction. After filtration, obtain the compound aqueous extract. Step 2: Add 1g of stachyose to the above compound aqueous extract, adjust the pH to 6.8, inoculate 4.1mL of Limosilactobacillus fermentum grx08 bacterial culture, and add a signal peptide with a final concentration of 0.2mmol / L. Step 3: Ferment at 37°C for 49.50 hours in an anaerobic environment, centrifuge, and collect the supernatant to obtain the fermentation composition.

[0018] The compound ingredients, including Astragalus membranaceus, lotus leaf, Poria cocos, and tangerine peel, are scientifically proportioned. Astragalus membranaceus tonifies Qi and raises Yang, lotus leaf clears heat and lowers lipids, Poria cocos strengthens the spleen and removes dampness, and tangerine peel regulates Qi and resolves phlegm, complementing each other to meet the needs of anti-obesity treatment. Using pure water as a solvent eliminates chemical pollution, allowing for the full release of active ingredients such as Astragalus membranaceus polysaccharides and lotus leaf flavonoids, while avoiding damage from heat-sensitive substances. Stachyose, as a prebiotic specific to probiotics, can directly reach the intestines and be selectively utilized by target strains, effectively promoting their proliferation and optimizing the intestinal microenvironment. Signal peptides can activate the expression of specific genes in the strains, inducing high production of antimicrobial peptides and active polysaccharides, forming a synergistic "nutrition-induction" effect with the strains. The anaerobic environment of 37℃ and pH value of 6.8 are precisely matched to the metabolic characteristics of Limosilactobacillus fermentum, combined with a suitable fermentation time of 49.5 hours, significantly improving fermentation efficiency. Finally, the supernatant is enriched by centrifugation to remove bacteria and residues, giving the composition the dual value of active ingredients and probiotic metabolites, with strong activity and synergistic effects.

[0019] In step one, the extraction temperature is 95℃ and the extraction time is 1.8 hours.

[0020] The 95℃ high temperature effectively disrupts the cross-linking structure of cellulose and pectin in plant cell walls, reduces the mechanical strength of cell walls, and promotes rapid cell wall rupture. This allows active ingredients that are originally difficult to dissolve in water, such as astragalus polysaccharides and lotus leaf flavonoids, to break free from their structural constraints and achieve full dissolution, significantly improving extraction efficiency. The 1.8-hour extraction time has been scientifically optimized to perfectly balance the dissolution efficiency and stability of the components. It avoids the problem of incomplete extraction of active ingredients due to excessively short extraction time, and also prevents the degradation of heat-sensitive substances such as vitexin caused by prolonged high temperatures, thus maximizing the preservation of the activity of effective components. The extract obtained under these parameters is pure and free of impurities, and is rich in nutrients such as carbohydrates and proteins required for the growth of probiotics. It can provide a high-quality substrate for subsequent fermentation, effectively ensuring the metabolic activity of the strains, creating favorable conditions for signal peptide-induced synergistic effects, and helping to maximize efficacy.

[0021] In step two, the bacterial concentration of Limosilactobacillus fermentum grx08 was 1×10⁻⁶. 8 -1×10 9 CFU / mL.

[0022] This concentration (1×10) 8 -1×10 9The concentration of CFU / mL is well-suited to the fermentation characteristics of the Limosilactobacillus fermentum strain. Combined with stachyose, it can rapidly proliferate, avoiding the lack of metabolites caused by insufficient viable cell count. At the same time, this concentration forms a highly efficient synergistic effect with the 0.2 mmol / L signal peptide. As a quorum sensing self-inducible peptide, the signal peptide can be recognized by the strain's two-component system, activating the transcription of specific genes and efficiently inducing the synthesis of target active substances such as antimicrobial peptides and active polysaccharides. This further enhances the efficacy of the composition in regulating lipid metabolism and balancing intestinal flora, highlighting the synergistic value of the fermentation process.

[0023] In step two, the amino acid sequence of the signal peptide is His-Gly-Glu-Gly-Thr-Phe-Ser-Ser-Glu. In the sequence, Gly gives the peptide chain good flexibility, the polar residues of Glu and Ser can maintain the structural stability under the fermentation environment of pH 6.5-7.0, and the hydrophobic fragment of Phe helps signal recognition and transmembrane transmission.

[0024] The flexibility of Gly ensures its efficient binding to the Limosilactobacillus fermentum grx08 two-component system receptor, while the polar residues of Glu and Ser ensure its structural stability in a pH environment of 6.5-7.0. The hydrophobic fragment of Phe accelerates signal recognition and transmembrane transmission. The three work synergistically to efficiently activate the quorum sensing system of the strain, inducing high production of functional metabolites such as antimicrobial peptides and active polysaccharides. This forms a synergistic closed loop with the active ingredients of the compound raw materials and stachyose, significantly improving the bioactivity and efficacy targeting of the fermentation composition.

[0025] In step three, the centrifugation speed is 8000 r / min and the centrifugation time is 15 minutes.

[0026] The high-speed operation of 8000 r / min generates a powerful centrifugal force of up to 7104 g, which can effectively overcome the diffusion effect of solid particles and accelerate the sedimentation process. This speed can accurately utilize density differences to achieve efficient separation of solid impurities of different particle sizes, such as Limosilactobacillus fermentum grx08 cells and compound raw material residues. Combined with a scientifically designed 15-minute centrifugation time, it avoids incomplete sedimentation of impurities due to excessively short time, while also preventing cell damage or secondary dissolution of impurities caused by excessively long centrifugation. The resulting supernatant is clear and transparent, free of turbidity and impurities, meeting the GMP requirements for controlling insoluble particles in pharmaceutical production. This provides high-quality raw materials for subsequent purification, fermentation, and other processes, ensuring product quality stability.

[0027] In step three, the fermentation composition was analyzed by UPLC-MS / MS. The results showed that the fermentation composition contained at least 53 substances with potential anti-obesity effects, and the content of astragaloside was increased by more than 30% compared with the unfermented compound aqueous extract. The lipase activity inhibition rate of the fermentation composition was ≥60%, and no hexanal or hexanol was detected.

[0028] UPLC-MS / MS offers high sensitivity and strong anti-interference capabilities, enabling precise qualitative and quantitative analysis and providing reliable scientific evidence for the advantages of the composite composition. 53 potential anti-obesity substances encompass the active ingredients of the raw materials and probiotic metabolites, forming a multi-target synergistic effect and enhancing the anti-obesity effect. The content of astragaloside is increased by more than 30%, and as a core component regulating lipid metabolism, it further enhances the targeted efficacy, fully demonstrating the technological value of signal peptide-induced fermentation. A lipase inhibition rate of ≥60% directly blocks key steps in fat absorption, verifying the core anti-obesity mechanism. No hexanal or hexanol (off-flavor substances from plant fermentation) were detected, ensuring the sensory quality and safety of the product and making it suitable for subsequent application scenarios.

[0029] Example 2: Verification of the anti-obesity effect of the fermentation composition (rat experiment) Experimental design: Sixty SPF-grade SD rats were randomly divided into 5 groups (n=12): normal diet group (ND), high-fat diet without intervention group (HFD), fermented compound group (SHLF08, administered by gavage at 10 mL / kg body weight of the product of Example 1), unfermented compound group (SHLa08, administered by gavage of unfermented water extract at the same dose), and Shenling Baizhu powder group (SBZ, administered by gavage of traditional Chinese medicine at the same dose). The intervention lasted for 8 weeks.

[0030] The advantage of this design is that it provides comprehensive grouping and sufficient control. The five groups form a complete control chain. The normal diet group (ND) serves as a blank control, clarifying the obesity-inducing effect of a high-fat diet. The high-fat diet without intervention group (HFD) validates the success of the model. The unfermented compound group (SHLa08) compares the efficacy difference between fermented and unfermented products, highlighting the synergistic value of fermentation. The Ginseng and Atractylodes Macrocephala Powder group (SBZ) serves as a positive control (known for improving metabolic syndrome), providing clinically relevant references for the results and eliminating the possibility of false positives. Regarding sample size and animal selection, SP... The study of metabolism and obesity using F-grade SD rats was conducted without interference from other bacteria, ensuring a high degree of standardization. The use of 12 rats per group (out of a total of 60) met statistical requirements, minimizing the impact of individual differences and satisfying the power requirements of tests such as analysis of variance, while also considering the reliability of the results and the ethical 3R principles. In terms of intervention and cycle design, a uniform gavage dose of 10 mL / kg ensured precise administration and avoided dosage deviations. The 8-week intervention cycle aligned with the changing patterns of obesity-related indicators (such as weight, blood lipids, and gene expression), effectively capturing efficacy differences and adapting to the metabolic regulatory characteristics of signal peptide-induced fermentation products.

[0031] result Body weight: The body weight of rats in the fermented compound group was 18.2% lower than that in the high-fat diet without intervention group, which was significantly higher than that in the unfermented compound group (9.5% lower) and the Shenling Baizhu powder group (10.3% lower). Blood lipids: Serum TG, TC, and LDL-C in the fermented food group were reduced by 32.5%, 28.1%, and 35.3% respectively compared to the high-fat diet without intervention group, while HDL-C increased by 27.6%. Gut microbiota: The abundance of Bifidobacterium in the fermented composition group was 2.3 times higher than that in the non-interventional group with a high-fat diet.

[0032] The weight loss (18.2%) was significantly higher than that of the unfermented compound group (9.5%) and the Shenling Baizhu powder group (10.3%), which clearly highlighted the synergistic effect of signal peptide-induced fermentation. All four blood lipid indicators were improved, significantly reducing "bad cholesterol" (LDL-C) and triglycerides (TG) and increasing "good cholesterol" (HDL-C), significantly reducing the risk of obesity-related cardiovascular diseases. The abundance of Bifidobacterium increased by 2.3 times. This species can regulate intestinal homeostasis and promote lipid metabolism, further confirming that the composition exerts its anti-obesity effect by improving the intestinal flora, and its mechanism is scientific and traceable.

[0033] Example 3: Human trial validation of the fermentation composition Experimental design: 120 overweight / obese college students aged 18-22 (BMI 24-28 kg / m²) were selected. 2 The participants were randomly divided into an experimental group (n=60, who took 100 mL of the fermented composition of Example 1 twice a day) and a control group (n=60, who took an equal amount of physiological saline) for 8 weeks.

[0034] University students aged 18-22 have uniform baseline characteristics, with a BMI of 24-28 kg / m². 2 It meets my country's overweight / obesity standards, effectively reducing the influence of confounding factors such as age and underlying diseases, resulting in higher compliance. Randomization ensures baseline balance between the two groups, while the control group receives an equal volume of saline, eliminating interference from factors such as natural weight loss and accurately attributing the efficacy to the fermented composition. With 60 participants in each group, totaling 120, it meets statistical power requirements and avoids false negatives. The dosage of 100mL twice daily and the 8-week cycle align with the changing patterns of obesity-related indicators, effectively capturing efficacy differences.

[0035] result Weight and body fat: In the experimental group, men lost 2.59 kg of weight and 2.13 kg of body fat, while women lost 3.29 kg of weight and 1.85 kg of body fat. There were no significant changes in the control group. Improvement in physical condition: Female participants showed a decrease in the conversion scores for Qi deficiency, phlegm-dampness, and damp-heat constitutions by 15.2, 18.7, and 14.3 points, respectively (out of 100). Safety: Serum ALT, AST, TBil, and creatinine levels in subjects remained within the normal reference range during the trial.

[0036] Both male and female participants experienced significant reductions in weight and body fat, while the control group showed no change. This clearly demonstrates that the composition's weight-loss effect is direct and targeted, unaffected by natural weight-loss factors. Women showed a significant reduction in the conversion scores for Qi deficiency, phlegm-dampness, and damp-heat constitutions, achieving a dual effect of weight loss and TCM constitution regulation, further expanding its applicable scenarios and value. Serum liver and kidney function indicators were all within the normal range, confirming the high safety of long-term use with no adverse reactions, providing a crucial guarantee for the industrial application of this composition.

[0037] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0038] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A signal peptide-induced fermentation composition based on, characterized in that, Prepared by the following steps: Step one: Huangqi, Heye, Fuling, and Chenpi were mixed in a mass ratio of (3-5):(2-4):(2-3):(1-2) to prepare a compound raw material. Deionized water was added to the compound raw material in an amount of 10 times the mass of the compound raw material, and the mixture was extracted. After filtration, a compound water extract was obtained. Step two: 1% stachyose was added to the compound water extract, the pH value was adjusted to 6.5-7.0, 4.1% Limosilactobacillus fermentum grx08 bacterial liquid was inoculated, and a signal peptide was added to a final concentration of 0.1-0.3 mmol / L. Step three: The mixture was fermented at 37°C in an anaerobic environment for 49.50 hours. After centrifugation, the supernatant was obtained, and a fermentation composition was obtained.

2. A signal peptide-induced fermentation composition according to claim 1, characterized in that: The temperature range for the extraction in step one is 90-100°C, and the extraction time range is 1.5-2 hours.

3. A signal peptide-induced fermentation composition according to claim 1, wherein: The bacterial concentration of the Limosilactobacillus fermentum grx08 bacterial solution in step two is 1 x 10 8 CFU / mL. 9 CFU / mL.

4. The signal peptide-induced fermentation composition of claim 1, wherein: The amino acid sequence of the signal peptide in step two is His-Gly-Glu-Gly-Thr-Phe-Ser-Ser-Glu. Gly provides good flexibility to the peptide chain, the polar residues of Glu and Ser can maintain the structural stability under the fermentation environment of pH 6.5-7.0, and the hydrophobic fragment of Phe helps signal recognition and transmembrane transmission.

5. The signal peptide-induced fermentation composition of claim 1, wherein: The centrifugation speed in step three is 8000 r / min, and the centrifugation time is 15 minutes.

6. The signal peptide-induced fermentation composition of claim 1, wherein: The fermentation composition in step three was detected by UPLC-MS / MS. The results showed that the fermentation composition contained at least 53 potential substances with anti-obesity effects, and the content of astragaloside in the fermentation composition was increased by more than 30% compared with the unfermented compound water extract. The lipase activity inhibition rate of the fermentation composition was ≥60%, and no n-hexanal or n-hexanol was detected.

7. Use of a fermentation composition induced by a signal peptide according to any one of claims 1-6 in the preparation of an anti-obesity product.

8. Use according to claim 7, characterized in that: The anti-obesity product includes an oral liquid, a solid beverage, a pressed candy, or a capsule.

9. Use according to claim 7, characterized in that: The anti-obesity product is used to reduce body weight, reduce body fat, improve dyslipidemia, or regulate TCM qi deficiency, phlegm-dampness, and damp-heat constitutions.

10. A signal peptide for use in inducing Limosilactobacillus fermentum grx08 in a fermentation of a pharmafood compound, characterized in that, The signal peptide is used to improve fermentation efficiency and the amount of anti-obesity active substances produced.